Mammalian CDP-diacylglycerol synthase

ABSTRACT

There is disclosed cDNA sequences and polypeptides having the enzyme CDP-diacylglycerol synthase (CDS) activity. CDS is also known as CTP:phosphatidate cytidylyltransferase. There is further disclosed methods for isolation and production of polypeptides involved in phosphatidic acid metabolism and signaling in mammalian cells, in particular, the production of purified forms of CDS.

TECHNICAL FIELD OF THE INVENTION

[0001] This present invention provides cDNA sequences and polypeptideshaving the enzyme CDP-diacylglycerol synthase (CDS) activity. CDS isalso known as CTP:phosphatidate cytidyltransferase (EC2.7.7.41). Thepresent invention further provides for isolation and production ofpolypeptides involved in phosphatidic acid metabolism and signaling inmammalian cells, in particular, the production of purified forms of CDS.

BACKGROUND OF THE INVENTION

[0002] CDP-diacylglycerol (DAG) is an important branch pointintermediate just downstream of phosphatidic acid (PA) in the pathwaysfor biosynthesis of glycerophosphate-based phospholipids (Kent, Anal.Rev. Biochem. 64: 315-343, 1995). In eukaryotic cells, PA, the precursormolecule for all glycerophospholipid, is converted either to CDP-DAG byCDP-DAG synthase (CDS) or to DAG by a phosphohydrolase. In mammaliancells, CDP-DAG is the precursor to phosphatidylinositol (PI),phosphatidylglycerol (PG), and cardiolipin (CL). Diacylglycerol is theprecursor to triacylglycerol, phosphatidylethanolamine, andphosphatidylcholine in eukaryotic cells. Therefore, the partitioning ofphosphatidic acid between CDP-diacylglycerol and diacylglycerol must bean important regulatory point in eukaryotic phospholipid metabolism(Shen et al., J. Biol. Chem. 271:789-795, 1996). In eukaryotic cells,CDP-diacylglycerol is required in the mitochondria forphosphatidylglycerol and cardiolipin synthesis and in the endoplasmicreticulum and possibly other organelles for the synthesis ofphosphatidylinositol (PI). PI, in turn, is the precursor for thesynthesis of a series of lipid second messengers, such asphosphatidylinositol-4,5-bisphosphate (PIP₂), DAG andinositol-1,4,5-trisphosphate (IP₃). Specifically, PIP₂ is the substratefor phospholipase C that is activated in response to a wide variety ofextracellular stimuli, leading to the generation of two lipid secondmessengers; namely, DAG for the activation of protein kinase C and IP₃for the release of Ca⁺⁺ from internal stores (Dowhan, Anal. Rev.Biochem. 66: 199-232, 1997).

[0003] The genes coding for CDS have been identified in E. coli (Icho etal, J. Biol. Chem. 260:12078-12083, 1985), in yeast (Shen et al., J.Biol. Chem. 271:789-795, 1996), and in Drosophila (Wu et al., Nature373:216-222, 1995). A human cDNA coding for CDS (hCDS1) is described byus herein and has been reported in Weeks et al., DNA Cell Biol. 16:281-289, 1997. Moreover, Heacock et al., J. Neurochem. 67: 2200-2203,1997 report cloning of a CDS1 from a human neuronal cell line.Furthermore, Lykidis et al., J. Biol. Chem 272:33402-33409 ,1997 andHalford et al., Genomics 54:140-144, 1998 both report DNA sequencessuspected to encode a human cds2 protein, but these references fail todisclose either biological activity or an intact N-terminal region forthe putative proteins.

[0004] It is of interest to isolate polynucleotides coding for human CDSand express them in mammalian cells to determine the potential roles ofthis enzyme in cellular function and use this enzyme as a target for thedevelopment of specific compounds that are modulators of its activity.With the advance in the understanding of disease processes, it has beenfound that many diseases result from the malfunction of intracellularsignaling. This recognition has led to research and development oftherapies based on the interception of signaling pathways in diseases(Levitzki, Curr. Opin. Cell Biol. 8:239-244, 1996). Compounds thatmodulate CDS activity, and hence modulate generation of a variety oflipid second messengers and signals involved in cell activation, aretherefore of therapeutic interest generally, and of particular interestin the areas of inflammation and oncology.

SUMMARY OF THE INVENTION

[0005] The present invention provides cDNA sequences, polypeptidesequences, and transformed cells for producing isolated recombinantmammalian CDS. The present invention provides two novel humanpolypeptides and fragment thereof, having CDS activity. The polypeptidesdiscovered herein are novel and will be called hCDS1 (human CDS1) andhCDS2 (human CDS2). CDS catalyzes the conversion of phosphatidic acid(PA) to CDP-diacylglycerol (CDP-DAG), which in turn is the precursor tophosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin(CL).

[0006] The present invention further provides nucleic acid sequencescoding for expression of the novel CDS polypeptides and active fragmentsthereof The invention further provides purified CDS mRNAs and antisenseoligonucleotides for modulation of expression of the genes coding forCDS polypeptides. Assays for screening test compounds for their abilityto modulate CDS activity are also provided.

[0007] Recombinant CDS is useful for screening candidate drug compoundsthat modulate CDS activity, particularly those compounds that activateor inhibit CDS activity. The present invention provides cDNA sequencesencoding a polypeptide having CDS activity and comprising the DNAsequence set forth in SEQ ID NO. 1 (hCDS1), the DNA sequence set forthin FIG. 8 (hCDS2), shortened fragments thereof, or additional cDNAsequences which due to the degeneracy of the genetic code encode apolypeptide of SEQ ID NO. 2 (hCDS1), a polypeptide of FIG. 8 (hCDS2), orbiologically active fragments thereof, or a sequence hybridizing theretounder high stringency conditions. The present invention further providesa polypeptide having CDS activity and comprising the amino acid sequenceof SEQ ID NO. 2 (hCDS1), the amino acid sequence of FIG. 8 (hCDS2), orbiologically active fragments thereof.

[0008] Also provided by the present invention are vectors containing aDNA sequence encoding a mammalian CDS enzyme in operative associationwith an expression control sequence. Host cells, transformed with suchvectors for use in producing recombinant CDS are also provided with thepresent invention. The inventive vectors and transformed cells areemployed in a process for producing recombinant mammalian CDS. In thisprocess, a cell line transformed with a cDNA sequence encoding a CDSenzyme in operative association with an expression control sequence, iscultured. The claimed process may employ a number of known cells as hostcells for expression of the CDS polypeptide, including, for example,mammalian cells, yeast cells, insect cells and bacterial cells.

[0009] Another aspect of this invention provides a method foridentifying a pharmaceutically-active compound by determining if aselected compound modulates the activity of CDS for converting PA toCDP-DAG. A compound having such activity is capable of modulatingsignaling kinase pathways and being a pharmaceutical compound useful foraugmenting trilineage hematopoiesis after cytoreductive therapy and foranti-inflammatory activity in inhibiting the inflammatory cascadefollowing hypoxia and reoxygenation injury (e.g., sepsis, trauma, ARDS,etc.).

[0010] The present invention further provides a transformed cell thatexpresses active mammalian CDS and further comprises a means fordetermining if a drug candidate compound is therapeutically active bymodulating recombinant CDS activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the cDNA sequence encoding hCDS1. The nucleotidesequence analysis and restriction mapping of the cDNA clone revealed a5′-untranslated region of 149 base pairs, an open reading frame capableof encoding a 461 amino acid polypeptide that spans nucleotide positions150 to 1535 and a 3′-untranslated region of 520 base pairs.

[0012]FIG. 2 shows the translated amino acid sequence of hCDS1.

[0013]FIG. 3 shows the amino acid sequence of hCDS1.

[0014]FIG. 4 shows the sequence homology among the hCDS1 codingsequence, the yeast CDS coding sequence, E. coli CDS coding sequence,and the Drosophila CDS coding sequence. This comparison shows that hCDS1has the greatest extended homology with amino acids 109 to 448 ofDrosophila CDS. The hCDS1 protein and the CDS protein from Drosophila,yeast, and E. coli have 45%, 21% and 7% overall match in amino acidsequence, respectively.

[0015]FIG. 5 shows the results of in vitro hCDS1 activity assays on cellfractions from stable transfectants of NCI-H460 cells. CDS activity wasassessed by conversion of (α-³²P)CTP to (³²P)CDP-DAG in in vitroreactions that required addition of an exogenous PA substrate. This is arepresentative histogram comparing the radiolabel incorporated intovarious cell fractions (membranes, cytosol, and nuclei/unbroken cells)from NCI-H460 cells stably transfected with the hCDS1 cDNA (pCE2.hCDS)or vector only (pCE2). In all fractions, the hCDS1 cDNA increasedradiolabel in the organic phase of the reactions. Total CDS activity wasmuch greater in membrane fractions, as would be expected for membraneassociated CDS, compared to cytosol fractions. Activity in unbrokencells masked the activity specific to nuclei.

[0016]FIG. 6 is a representative phosphorimage of [³²P]phospholipidsfrom membrane fraction CDS assay reactions after the second dimension offfTLC. FIG. 6 confirms that the radiolabeled product found in themembrane fractions does migrate with a CDP-DAG standard on TLC. Theidentities of labeled bands were determined by migration of phospholipidstandards visualized by UV or FL imaging on the STORM after primulinstaining. Lanes 1-3 represent triplicate samples derived from membranesof NCI-H460 cells transfected with the hCDS1 expression vector, andlanes 4-6 represent triplicate samples from transfectants with thecontrol vector. Cells transfected with the hCDS1 cDNA showed 1.6-2.4fold more CDS activity in membrane fractions than vector transfectants.The relative CDS activity between hCDS1 transfectants and vectortransfectants was similar when determined by scintillation counting orTLC analysis. These data indicate that the hCDS1 cDNA clone of SEQ IDNO. 1 does encode CDS activity.

[0017]FIGS. 7A and 7B show, respectively, that production of TNF-α(tumor necrosis factor alpha) and IL-6 in ECV304 cells stablytransfected with a hCDS1 expression vector increases by greater thanfive fold relative to ECV304 cells stably transfected with controlvector after equal stimulation with IL-1β (interleukin-1 beta). Therewas little effect on basal level of cytokine release. These dataindicate that overexpression of hCDS1 amplified the cytokine signalingresponse in these cells, as opposed to enhancing steady state, basalsignals.

[0018]FIG. 8 shows the DNA and amino acid sequence of hCDS2.

[0019]FIG. 9 shows an amino acid sequence alignment of the hCDS2 codingsequence with the hCDS1 coding sequence. The amino acids that areidentical between the two sequences are highlighted.

[0020]FIG. 10 shows the results of a TLS analysis of hCDS2 production of[32P]CDP-DAG after TLC analysis

[0021]FIG. 11 shows expression of hCDS1 and hCDS2 mRNAs in cancer versusnormal prostate tissues.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides novel, isolated, biologicallyactive mammalian CDS enzymes. The term “isolated” means any CDSpolypeptide of the present invention, or any other gene encoding CDSpolypeptide, which is essentially free of other polypeptides or genes,respectively, or of other contaminants with which the CDS polypeptide orgene might normally be found in nature.

[0023] The invention includes a biologically active polypeptide, CDS,and biologically active fragments thereof. As used herein, the term“biologically active polypeptide” refers to a polypeptide whichpossesses a biological function or activity which is identified througha biological assay, preferably cell-based, and which results in theformation of CDS-DAG species from PA. A “biologically activepolynucleotide” denotes a polynucleotide which encodes a biologicallyactive polypeptide. The term “biologically active fragment,” as usedherein, refers to a nucleotide or polypeptide sequence in which one ormore amino acids or nucleotides has been deleted but which retains CDSactivity.

[0024] Minor modification of the CDS primary amino acid sequence mayresult in proteins which have substantially equivalent activity ascompared to the sequenced CDS polypeptide described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the polypeptides produced by these modificationsare included herein as long as the activity of CDS is present. This canlead to the development of a smaller active molecule which would havebroader utility. For example, the present invention includes removal ofone or more amino, carboxy terminal, or internal amino acids from theCDS polypeptide, so long as such amino acids are not required for CDSactivity.

[0025] The CDS polypeptide of the present invention also includesconservative variations of the polypeptide sequence. The term“conservative variation” denotes the replacement of an amino acidresidue by another, biologically active similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue, such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofparent amino acid provided that antibodies raised to the substitutedpolypeptide also immunologically react with the unsubstitutedpolypeptide.

[0026] The present invention further includes allelic variations(naturally-occurring base changes in the species population which may ormay not result in an amino acid change) of the DNA sequences hereinencoding active CDS polypeptides and active fragments thereof.

[0027] The inventive DNA sequences further comprise those sequenceswhich hybridize under high stringency conditions (see, for example,Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold SpringHarbor Laboratory, pages 387-389, 1982) to the coding region of hCDS1(e.g., nucleotide #150 to nucleotide #1535 in SEQ ID NO. 1) or thecoding region of hCDS2 (FIG. 8) and which have CDS activity. Highstringency conditions include 5× SSC at 65° C., followed by washing in0.1× SSC at 65° C. for thirty minutes or, alternatively, 50% formamide,5× SSC at 42° C.

[0028] The present invention also includes nucleotide sequences havingat least an 85%, at least a 90%, or at least a 95% sequence identity tothe nucleotide sequence of the coding region of hCDS2 (FIG. 8) and whichhave CDS activity. The present invention further includes a polypeptidehaving at least an 85%, at least a 90%, or at least a 95% sequenceidentity to the hCDS2 polypeptide shown in FIG. 8 and which have CDSactivity. As used herein, the term “sequence identity” denotes the“match percentage” calculated by the DNASIS computer program (Version2.5 for Windows; available from Hitachi Software Engineering Co., Ltd.,South San Francisco, Calif.) using standard defaults as described in thereference manual accompanying the software, which is incorporated hereinby reference.

[0029] With regard to the above-described fragments of hCDS2, sequencesthat hybridize to hCDS2, and sequences having sequence identity tohCDS2, the invention includes embodiments where these sequences have anintact CDS N-terminal region.

[0030] The present invention further includes DNA sequences which codefor CDS polypeptides having CDS activity but differ in codon sequencedue to degeneracy of the genetic code. Variations in the DNA sequenceswhich are caused by point mutations or by induced modifications of thesequence of SEQ ID NO. 1 or FIG. 8, which enhance the activity of theencoded polypeptide or production of the encoded CDS polypeptide arealso encompassed by the present invention.

[0031] CDS Sequence Discovery

[0032] hCDS1

[0033] A homology search of the Genbank database (Boguski, et al.,Science 265:1993-1994, 1994) of expressed sequence tags (dbEST) usingDrosophila CDS protein sequence as a probe came up with several shortstretches of cDNA sequence with homology to the Drosophila CDS proteinsequence. These cDNA sequences were derived from single-run partialsequencing of random human cDNA clones carried out mainly by I.M.A.G.E.Consortium [LLNL] cDNA clones program. An example of the amino acidsequence homology between the Drosophila CDS and a human cDNA clone(IMAGE Clone ID #135630) is shown below: 371KRAFKIKDFGDMIPGHGGIMDRFDCQFLMATFVNVYIS 408     KRAFKIKDF+ IPGHGGIMDRFDCQ+LMATFV+VYI+  11 KRAFKIKDFANTIPGHGGIMDRFDCQYLMATFVHVYIT124

[0034] The top line (SEQ ID NO. 3) refers to the Drosophila CDS sequencefrom amino acids 371 to 408 and the bottom line (SEQ ID NO. 4) refers toa homologous region from IMAGE Clone ID #135630 translated using readingframe +2. Identical amino acids between these two sequences are shown onthe middle line with the “+” signs indicating conservative amino acidchanges. In order to determine if such cDNA clones with this level ofhomology to the Drosophila CDS sequence encoded human CDS sequence, itwas necessary to isolate the full-length cDNA clone, insert it into anexpression vector, and test if cells transfected with the cDNAexpression vector will produce more CDS activity.

[0035] Accordingly, a synthetic oligonucleotide (o.h.cds.1R), 5′-CCCACCATGG CCAGGAATGG TATTTGC-3′ (SEQ ID NO. 5), was made based on thecomplement sequence of the amino acid region, ANTIPGHGG, of IMAGE CloneID #135630 for the isolation of a putative human cDNA clone from aSuperScript human leukocyte cDNA library (Life Technologies,Gaithersburg, Md.) using the GeneTrapper cDNA positive selection system(Life Technologies, Gaithersburg, Md.). The colonies obtained frompositive selection were screened with a [γ-³²P]-ATP labeled syntheticoligonucleotide (o.h.cds.1), 5′-AGTGATGTGA ATTCCTTCGT GACAG-3′ (SEQ IDNO. 6), corresponding to nucleotides 144-168 of IMAGE Clone ID #133825.Of the few cDNA clones that hybridized with the o.h.cds.1 probe, cloneLK64 contained the largest cDNA insert with a size of 1700 base pairs.DNA sequence analysis of LK64 showed the translated sequence of itslargest open reading frame from the 5′ end contained extensive homologywith amino acids 109 to 448 of the Drosophila CDS protein sequence.Clone LK64 did not appear to contain a full-length cDNA insert for CDS.It was missing the coding region corresponding to the first 110 aminoacids from the N-terminus. A second homology search of the Genbankdatabase (Boguski, et al., Science 265:1993-1994, 1994) using the3′-untranslated sequence of LK64 as a probe came up with more shortstretches of cDNA sequences with perfect homology to the 3′ end of theputative human CDS clone LK64. Restriction mapping and DNA sequenceanalysis of IMAGE Clone ID #145253 (Genome Systems, St. Louis, Mo.),derived from a placental cDNA library, showed it contained extensivesequence homology with the N-terminal coding region of the DrosophilaCDS and overlapped with the sequence obtained from clone LK64.

[0036] To assemble the putative full-length human CDS cDNA clone, a 500base pair Pst I-Nco I fragment from of IMAGE Clone ID #145253 and a 1500base pair Nco I -Not I fragment from LK64 were isolated. These twofragments were inserted into a Pst I and Not I digested vectorpBluescriptII SK(−) vector via a three-part ligation to generatepSK.hcds.

[0037]FIG. 1 shows the cDNA sequence of hCDS1. The nucleotide sequenceanalysis and restriction mapping of the cDNA clone revealed a5′-untranslated region of 149 base pairs, an open reading frame encodinga 461 amino acids polypeptide that spans nucleotide positions 150 to1535 and a 3′-untranslated region of 520 base pairs (FIG. 2). The ATGinitiation site for translation was identified at nucleotide positions150-152 and fulfilled the requirement for an adequate initiation site.(Kozak, Critical Rev. Biochem. Mol. Biol. 27:385-402, 1992). There wasanother upstream ATG at positions 4-6 but it was followed by an in-phasestop codon at positions 19-20. The calculated molecular weight of hCDS1is 53,226 daltons with a predicted pI of 7.57.

[0038] The sequence of the 461 amino acid open reading frame (FIG. 3)was used as the query sequence to search for homologous sequences inprotein databases. A search of Genbank Release 92 from the NationalCenter for Biotechnology Information (NCBI) using the BLAST programshowed that this protein was most homologous to the Drosophila CDS, theyeast CDS, and the E. coli CDS. FIG. 4 shows amino acid sequencealignment of this putative human CDS coding sequence with the DrosophilaCDS, the yeast CDS, and the E. coli coding sequences, showing that thehuman CDS is most homologous to the Drosophila CDS.

[0039] hCDS2

[0040] A homology search of the Genbank database (Boguski, et al.,Science 265:1993-1994, 1994) of expressed sequence tags (dbEST) usingthe hCDS1 protein sequence (Weeks et al, DNA Cell Biol. 16: 281-289,1997) as probe came up with several short stretches of human cDNAsequences that were homologous but distinct from the hCDS1 sequence.These cDNA sequences were derived from single-run partial sequencing ofrandom human cDNA clones projects carried out mainly by I.M.A.G.E.Consortium [LLNL] cDNA clones program.

[0041] Of these sequences, IMAGE Clone ID#485825 was found to have thefollowing homology to the coding region of hCDS1 from amino acids227-271:         10        20        30        40QSHLVIHNLFEGMIWFIVPISCVICNDIMAYMFGFFFGRTPLIKLX:::::.:::::::::.::::.:::::: ::.:::::::::::::QSHLVTQNLFEGMIWFLVPISSVICNDITAYLFGFFFGRTPLIKL 230       240       250       260       270

[0042] The top line refers to IMAGE Clone ID#485825 translated usingreading frame +3 and the bottom line refers to the coding region ofhCDS1 from amino acids 227-271. Identical amino acids between these twosequences are shown on the middle line as “:” and with the “.” signsindicating conservative amino acid changes. Since the 5′-end of the cDNAinsert of IMAGE Clone ID#485825 corresponded to amino acid 227 of hCDS1,this clone therefore does not appear to contain a full-length cDNAinsert for CDS, most likely missing the coding region corresponding tothe first 220 amino acids from the N-terminus. A second homology searchof the Genbank database (Boguski, et al., Science 265:1993-1994, 1994)of expressed sequence tags (dbEST) using the sequence of IMAGE CloneID#485825 as probe came up with a clone with a longer cDNA insert (cloneID#663789) from the Genbank database with perfect homology to the IMAGEClone ID#485825. Restriction mapping and DNA sequence analysis of IMAGEClone ID#663789 (Genome Systems, St. Louis, Mo.) showed it to be alonger cDNA clone with extensive sequence homology with the codingregion of hCDS1 but still missing the first 60 amino acids in the codingregion. To isolate the 5′-coding region of hCDS2 cDNA, a syntheticoligonucleotide, 5′- AGGACGCATA TGAGTGGTAG AC-3′ (oCDS2_(—)2R),complementary to a region spanning the Nde I site near the 5′ portion ofclone ID#663789 was used in combination with a forward vector primer(o.sport.1), 5′- GACTCTAGCC TAGGCTTTTG C-3′ for amplification of the5′-region from a pCMV.SPORT human leukocyte cDNA library (LifeTechnologies, Gaithersburg, Md.). PCR fragments generated that were >400bp were inserted into the pGEM-T vector (Promega, Madison, Wis.) forfurther analysis. Restriction mapping and DNA sequence analysis showedone of the clones, pCDS2.H7, to be homologous to the N-terminal codingregion of hCDS1.

[0043] To assemble the putative full-length human CDS cDNA clone, the420 bp Acc65 I-Nde I fragment from pCDS2.H7 and the 1200 bp Nde I-Xba Ifragment from clone ID#663789 were isolated. These two fragments wereinserted into a Acc65 I and Xba I digested vector pBluescript SK(−)IIvector via a three-part ligation to generate pSK.CDS2.

[0044]FIG. 8 shows the DNA sequence ID of the hCDS2. The nucleotidesequence analysis and restriction mapping of the cDNA clone revealed a5′-untranslated region of 24 bp, an open reading frame capable ofencoding a 445 amino acid polypeptide that spans nucleotide positions 25to 1362 and a 3′-untranslated region of 1126 bp. The ATG initiation sitefor translation was localized at nucleotide positions 25-27 andfulfilled the requirement for an adequate initiation site according toKozak (Kozak, Critical Rev. Biochem. Mol. Biol. 27:385-402, 1992).

[0045] Amino acid sequence alignment of the hCDS2 coding sequence withthe human CDS1 shows 64% identity (FIG. 9). The amino acids that areidentical between the two sequences are highlighted.

[0046] Expression of human CDS cDNA in mammalian cells

[0047] hCDS1

[0048] To see if overexpression of hCDS1 would have any effect onmammalian cells, the entire cDNA insert (˜2,000 base pairs) frompSK.hcds was cleaved with Asp718 I and Not I for insertion into themammalian expression vector pCE2 to generate pCE2.hCDS. The plasmid pCE2was derived from pREP7b (Leung et al. Proc. Natl. Acad. Sci. USA,92:4813-4817, 1995) with the RSV promoter region replaced by the CMVenhancer and the elongation factor-1α (EF-1α) promoter and intron. TheCMV enhancer came from a 380 base pair Xba I-Sph I fragment produced byPCR from pCEP4 (Invitrogen, San Diego, Calif.) using the primers5′-GGCTCTAGAT ATTAATAGTA ATCAATTAC-3′ (SEQ ID NO. 7) and 5′-CCTCACGCATGCACCATGGT AATAGC-3′ (SEQ ID NO. 8). The EF-1α promoter and intron(Uetsuki et al., J. Biol. Chem., 264:5791-5798, 1989) came from a 1200base pair Sph I-Asp718 I fragment produced by PCR from human genomic DNAusing the primers 5′-GGTGCATGCG TGAGGCTCCG GTGC-3′ (SEQ ID NO. 9) and5′-GTAGTTTTCA CGGTACCTGA AATGGAAG-3′ (SEQ ID NO. 10). These 2 fragmentswere ligated into a Xba I/Asp718 I digested vector derived from pREP7bto generate pCE2.

[0049] A second clone, pCE2.hCDS2, was constructed that lacked the humanCDS 3′-UT region (520 nt). An Asp718 I (in the multiple cloningsite)/NcoI fragment and a NcoI/BamHI fragment from pSK.hCDS werecombined in a three-part ligation with Asp718 I/BamHI digested pCE2.Northern blot analysis of 293-EBNA human embryonic kidney cellstransiently transfected with CDS cDNA expression plasmids (pCE2.hCDS orpCE2.hCDS2) showed that deletion of the entire 3′-UT region had littleeffect on CDS steady-state mRNA levels.

[0050] The CDS activity in transfected cell fractions (membranes,cytosol, nuclei/unbroken cells) was determined by incorporation of(α-³²P)CTP into (³²P)CDP-DAG in the presence of exogenously added PAsubstrate. Cells were fractionated by resuspending previously frozencell pellets in cold hypotonic lysis buffer (HLB; 10 mM KCl, 1.5 mMMgCl₂, 10 mM Tris, pH 7.4, 2 mM benzamidine HCl, and 10 μg/ml eachleupeptin, soybean trypsin inhibitor, and pepstatin A) at approx. 5×10⁷cells/ml. After 10 min. on ice, cells were dounced (Wheaton pestle A) 40strokes, then spun 500 ×g, 10 min. at 4° C. to remove nuclei andunbroken cells. The resuspension of the pellet, incubation, and lowspeed spin were repeated twice. The final “nuclei/unbroken cells” pelletwas resuspended in 50-100 μl HLB. Supernatants were spun at 109,000× g,30 min. at 4° C. generating “cytosol” supernates and “membrane” pellets.The pellets were resuspended in 150-225 μl HLB. An aliquot of eachfraction was removed for determination of protein concentration by a BCAassay. Fractions were stored at −70° C. All assays were done onfractions after one thaw.

[0051] The in vitro CDS activity assay conditions were a modification ofmethods described previously (Mok et al., FEBS Letters 312:236-240,1992;and Wu et al., Nature 373:216-222,1995). Briefly, each 0.3 ml reactioncombined 0.23 mM PA (Sigma; from egg yolk lecithin), 50 mM Tris-maleate,pH 7.0, 1.5% Triton X-100, 0.5 mM DTT, 75-500 μg protein from cellfractions, 30 mM MgCl₂, and 2 μCi (α-³²P)CTP. MgCl₂ and (α-³²P)CTP wereadded just prior to a 10 min. incubation at 37° C. The reactions wereterminated with 4 ml chloroform:methanol (1:1) and vortexing. Theorganic phase was extracted three times with 1.8 ml 0.1 N HCl with 1 MNaCl, and vortexing. Radioactivity in the organic phase was determinedby scintillation counting or TLC.

[0052] A flip-flop TLC (ffTLC) system (Gruchalla et al., J. Immunol.144:2334-2342, 1990) was modified for the separation of CDP-DAG and PA.Specifically, 200 ml of organic phase was dried and brought up in 20 μLCHCl₃:MeOH (2:1) and spotted in the center of a 20×20 cm TLC plate(Analtech Silica Gel HP-HLF). TLC was run in CHCl₃:MeOH:NH₄OH:H₂O(65:30:4:1) until the solvent had reached the top of the plate. In thissolvent system, neutral and cationic lipids migrate, whereas PA, CDP-DAGand other anionic lipids stay near the origin. The plate was dried andvisualized by UV with 0.05% primulin stain (Sigma, St. Louis, Mo.) in80% acetone. The plate was cut below the PC standard, and the bottomhalf of the plate was rotated 180° and run in CHCl₃:MeOH:Acetic Acid:H₂O(80:25:15:5) to enable migration of the anionic lipids until the solventreached the top of the plate. The radioactive bands on the TLC platewere quantified using a STORM® phosphorimager (Molecular Dynamics,Sunnyvale, Calif.). Non-radiolabeled lipid standards were stained withprimulin and visualized by fluorescence using the STORM®.

[0053]FIG. 5 shows the results of in vitro CDS activity assays on cellfractions from stable transfectants of NCI-H460 cells. CDS activity wasassessed by conversion of (α-³²P)CTP to (³²P)CDP-DAG in in vitroreactions that required addition of an exogenous PA substrate. This is arepresentative histogram comparing the radiolabel incorporated intovarious cell fractions (membranes, cytosol, and nuclei/unbroken cells)from NCI-H460 cells stably transfected with the hCDS1 cDNA (pCE2.hCDS)or vector only (pCE2). In all fractions, the CDS cDNA increasedradiolabel in the organic phase of the reactions. Total CDS activity wasmuch greater in membrane fractions, as would be expected for membraneassociated CDS, compared to cytosol fractions. Activity in unbrokencells masked the activity specific to nuclei.

[0054]FIG. 6 is a representative phosphorimage of [³²P]phospholipidsfrom membrane fraction CDS assay reactions after the second dimension offfTLC. FIG. 6 confirms that the radiolabeled product found in themembrane fractions does migrate with a CDP-DAG standard on TLC. Theidentities of labeled bands were determined by migration of phospholipidstandards visualized by UV or FL imaging on the STORM after primulinstaining. Lanes 1-3 represent triplicate samples derived from membranesof NCI-H460 cells transfected with the hCDS1 expression vector, andlanes 4-6 represent triplicate samples from transfectants with thecontrol vector. Cells transfected with the hCDS1 cDNA showed 1.6-2.4fold more CDS activity in membrane fractions than vector transfectants.The relative CDS activity between CDS transfectants and vectortransfectants was similar when determined by scintillation counting orTLC analysis. Similar CDS activity was seen in two different transfectedhuman cell lines, NCI-H460 and ECV304. The average specific activity ofCDS in membranes of CDS transfectants was 2.7 fmol/min/mg proteincompared to 1.4 fmol/min/mg protein in membranes of vectortransfectants. These results demonstrated that overexpression of thehuman CDS cDNA clone lead to an increase in CDS activity in cellfractions and that activity in an in vitro assay was completelydependent on the addition of PA. These data indicate that the human cDNAclone of SEQ ID NO. 1 does encode CDS activity.

[0055] hCDS2

[0056] To see if overexpression of hCDS2 has an effect in mammaliancells, the entire cDNA insert (˜1,900 bp) from pSK.CDS2 was cleaved withAsp718 I and Xba I for insertion into a mammalian inducible expressionvector pIND (Invitrogen, San Diego, Calif.) to generate pI_CDS2.

[0057] pI_CDS2 DNA and pVgRXR (Invitrogen, San Diego, Calif.) DNA wereco-transfected into ECV304 cells (American Type Culture Collection,Rockville, Md.) with a Cell-Porator™ (Life Technologies, Gaithersburg,Md.) using conditions described previously (Cachianes, et al.,Biotechniques 15:255-259, 1993). After adherence of the transfectedcells 24 hours later, the cells were grown in the presence of 500 μg/mlG418 (Life Technologies, Gaithersburg, Md.) and 100 μg/ml Zeocin(Invitrogen, San Diego, Calif.) to select for cells that hadincorporated both plasmids. G418 and Zeocin resistant clones thatexpressed CDS2 mRNA at a level more than 10 fold higher in the presenceof muristerone A (Invitrogen, San Diego, Calif.) relative to uninducedor untranfected cells based on Northern Blot analysis (Kroczek, et al.,Anal. Biochem. 184: 90-95, 1990) were selected for further study.

[0058] The CDS activity in ECV304 cells transfected with pI_CDS2 DNA andpVgRXR DNA with or without muristerone A induction was compared using aTLC assay (Weeks et al, DNA Cell Biol. 16: 281-289, 1997).

[0059]FIG. 10 shows an example of hCDS2 assay results by measuring theproduction of [32P]CDP-DAG after TLC analysis. The identities of labeledbands were determined based on Rf values obtained for standardphospholipids visualized by primulin staining. The left two barsrepresent triplicate samples derived from ECV304 cells transfected withpVgRXR and the control vector pIND in the absence or presence of theinducer muristerone A. The enzyme activity found here representsendogenous CDS activity found in ECV304 cells, as cells without or withmuristerone A treatment produced similar activity. The right two barsrepresent triplicate samples derived from ECV304 cells transfected withpVgRXR and the inducible CDS2 vector pI_CDS2 in the absence or presenceof the inducer muristerone A. Quantitation of the radioactive bandscorresponding to CDP-DAG shows cells transfected with the induciblehCDS2 expression plasmid have an approximately two fold increase inactivity after induction with muristerone A compared to same cellswithout induction or to vector control cells either with or withoutinduction, showing that the hCDS2 cDNA clone encode a protein having CDSactivity.

[0060] Complementation of yeast cds1 mutant with hCDS1

[0061] As the yeast CDS gene is essential for growth (Shen et al., J.Biol. Chem. 271:789-795, 1996), another way to show that the cDNA doesencode CDS activity was to determine if the human CDS cDNA willcomplement the growth defect of a mutant yeast strain with a deletion inthe endogenous yeast CDS gene. Accordingly, the hCDS1 cDNA was cloneddownstream of a GAL1 promoter in a yeast expression vector.Specifically, a Hind III-Sac I fragment from pSK.hCDS was inserted intopYES.LEU vector to generate pYES.hCDS. pYES.LEU was derived from pYES2(Invitrogen, San Diego, Calif.) by inserting a BspH I fragmentcontaining a LEU2 marker from pRS315 (Sikorski et al., Genetics122:19-27, 1989) into the Nco I of pYES2. pYES.hCDS was introduced intoa null cds1 strain of yeast, YSD90A (Shen et al., J. Biol. Chem.271:789-795, 1996), with a covering plasmid, pSDG1, carrying thefunctional yeast CDS1. The latter plasmid was cured from cells by growthin media lacking leucine but containing uracil and galactose. PCRanalysis confirmed the absence of the yeast CDS1 gene and Northern blotanalysis verified expression of the hCDS1 cDNA. This strain was found tobe absolutely dependent on galactose for growth. Galactose activates theGAL1 promoter for the production of human CDS protein. When the carbonsource was switched to glucose, which would shut down the GAL1 promoter,growth stopped completely in less than a generation. These data show thehuman CDS was able to complement the growth defect of a yeast cds1mutant.

[0062] The cells grown on galactose were lysed and assayed for CDSactivity according to the assay method described (Shen et al., J. Biol.Chem. 271:789-795, 1996). The specific activity using yeast conditionsshowed activity at 20% of single copy CDS1 wild type activity. This isconsistent with the above plasmid in a wild type background showingapproximately 1.3 fold increase in activity when grown on galactoseversus glucose.

[0063] The following experiment found that hCDS1 over-expressionenhanced cytokine induced signaling in cells. Over-expression of CDS wasexpected to alter the cellular level of various lipid second messengerssuch as PA, IP₃ and DAG (Kent, Anal. Rev. Biochem. 64:315-343, 1995) andhence modulates cytokine induced signaling response in cells. To testthis hypothesis, a hCDS1 expression plasmid (pCE2.hCDS), or vector(pCE2) were stably transfected into ECV304 cells (American Type CultureCollection, Rockville, Md.), an endothelial cell line that produces IL-6and TNF-α upon stimulation with IL-1β. FIG. 7 shows that the secretionof TNF-α IL-6 in ECV304 cells stably transfected with CDS expressionvector increased by >5 fold relative to ECV304 cells stably transfectedwith control vector after stimulation with 1 ng/ml IL-1β. However, therewas little effect on the basal level of cytokine release, suggestingthat over-expression of CDS amplified the cytokine signaling response,as opposed to enhancing the steady-state, basal signal, in these cells.

[0064] Expression of hCDS1 and hCDS2 mRNA in cancer versus normalprostate tissue

[0065] To examine if CDS mRNA expression in cancer versus normaltissues, RT-PCR was performed on specimens of prostate cancer tissuesand the corresponding normal prostate tissues in the surgical marginsfrom four independent patients. FIG. 11 shows hCDS1 mRNA was elevated inprostate cancer in 2 out of 4 patients, whereas hCDS2 mRNA was elevatedin prostate cancer in 3 out of 4 patients. A housekeeping geneβ2-microglobulin mRNA level was found to be similar in normal and cancerprostate tissues. ETS-2, a transcription factor reported to be elevatedin prostate cancer (Liu et al., Prostate 30: 145-153, 1997), was foundto be elevated in the same 3 out of 4 patients examined here, suggestinghCDS2, like ETS-2, may be a target for drug intervention in cancertherapy.

[0066] CDS Polypeptide Synthesis

[0067] Polypeptides of the present invention can be synthesized by suchcommonly used methods as t-BOC or FMOC protection of alpha-amino groups.Both methods involve step-wise syntheses whereby a single amino acid isadded at each step starting from the C-terminus of the peptide (Coliganet al., Current Protocols in Immunology, Wiley Interscience, Unit 9,1991). In addition, polypeptides of the present invention can also besynthesized by solid phase synthesis methods (e.g., Merrifield, J. Am.Chem. Soc. 85:2149, 1962; and Steward and Young, Solid Phase PeptideSynthesis, Freeman, San Francisco pp. 27-62, 1969) using copolyol(styrene-divinylbenzene) containing 0.1-1.0 mM amines/g polymer. Oncompletion of chemical synthesis, the polypeptides can be deprotectedand cleaved from the polymer by treatment with liquid HF 10% anisole forabout 15-60 min at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution, which isthen lyophilized to yield crude material. This can normally be purifiedby such techniques as gel filtration of Sephadex G-15 using 5% aceticacid as a solvent. Lyophilization of appropriate fractions of the columnwill yield a homogeneous polypeptide or polypeptide derivatives, whichare characterized by such standard techniques as amino acid analysis,thin layer chromatography, high performance liquid chromatography,ultraviolet absorption spectroscopsy, molar rotation, solubility andquantitated by solid phase Edman degradation.

[0068] CDS Polynucleotides

[0069] The invention also provides polynucleotides which encode the CDSpolypeptide of the invention. As used herein, “polynucleotide” refers toa polymer of deoxyribonucleotides or ribonucleotides in the form of aseparate fragment or as a component of a larger construct. DNA encodingthe polypeptide of the invention can be assembled from cDNA fragments orfrom oligonucleotides which provide a synthetic gene which is capable ofbeing expressed in a recombinant transcriptional unit. Polynucleotidesequences of the invention include DNA, RNA and cDNA sequences.Preferably, the nucleotide sequence encoding CDS is the sequence of SEQID NO. 1 or of FIG. 8. DNA sequences of the present invention can beobtained by several methods. For example, the DNA can be isolated usinghybridization procedures which are known in the art. Such hybridizationprocedures include, for example, hybridization of probes to genomic orcDNA libraries to detect shared nucleotide sequences, antibody screeningof expression libraries to detect common antigenic epitopes or sharedstructural features and synthesis by the polymerase chain reaction(PCR). Such hybridization includes hybridization under high stringencyconditions as described above.

[0070] Hybridization procedures are useful for screening recombinantclones by using labeled mixed synthetic oligonucleotides probes, whereineach probe is potentially the complete complement of a specific DNAsequence in a hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful fordetection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. Using stringent hybridization conditions to avoid non-specificbinding, it is possible to allow an autoradiographic visualization of aspecific genomic DNA or cDNA clone by the hybridization of the targetDNA to a radiolabeled probe, which is its complement (Wallace et al.Nucl. Acid Res. 9:879, 1981). Specific DNA sequences encoding CDS canalso be obtained by isolation and cloning of double-stranded DNAsequences from the genomic DNA, chemical manufacture of a DNA sequenceto provide the necessary codons for the complete polypeptide of interestor portions of the sequence for use in PCR to obtain the completesequence, and in vitro synthesis of a double-stranded DNA sequence byreverse transcription of mRNA isolated from a eukaryotic donor cell. Inthe latter case, a double-stranded DNA complement of mRNA is eventuallyformed which is generally referred to as cDNA. Of these three methodsfor developing specific DNA sequences for use in recombinant procedures,the isolation of cDNA clones is the most useful. This is especially truewhen it is desirable to obtain the microbial expression of mammalianpolypeptides since the presence of introns in genomic DNA clones canprevent accurate expression.

[0071] The synthesis of DNA sequences is sometimes a method that ispreferred when the entire sequence of amino acids residues of thedesired polypeptide product is known When the entire sequence of aminoacid residues of the desired polypeptide is not known, direct synthesisof DNA sequences is not possible and it is desirable to synthesize cDNAsequences isolation can be done, for example, by formation of plasmid-or phage-carrying cDNA libraries which are derived from reversetranscription of mRNA. mRNA is abundant in donor cells that have highlevels of genetic expression. In the event of lower levels ofexpression, PCR techniques can be used to isolate and amplify the cDNAsequence of interest. Using synthesized oligonucleotides correspondingexactly, or with some degeneracy, to known CDS amino acid or nucleotidesequences, one can use PCR to obtain and clone the sequence between theoligonucleotides. The oligonucleotide may represent invariant regions ofthe CDS sequence and PCR may identify sequences (isoforms) withvariations from SEQ ID NO. 1 or FIG. 8.

[0072] A cDNA expression library, such as lambda gt11, can be screenedindirectly for the CDS polypeptide, using antibodies specific for CDS.Such antibodies can be either polyclonal or monoclonal, derived from theentire CDS protein or fragments thereof, and used to detect and isolateexpressed proteins indicative of the presence of CDS cDNA.

[0073] A polynucleotide sequence can be deduced from an amino acidsequence by using the genetic code, however the degeneracy of the codemust be taken into account. Polynucleotides of this invention includevariant polynucleotide sequences which code for the same amino acids asa result of degeneracy in the genetic code. There are 20 natural aminoacids, most of which are specified by more that one codon (a three basesequence). Therefore, as long as the amino acid sequence of CDS resultsin a biologically active polypeptide (at least, in the case of the sensepolynucleotide strand), all degenerate nucleotide sequences are includedin the invention. The polynucleotide sequence for CDS also includessequences complementary to the polynucleotides encoding CDS (antisensesequences). Antisense nucleic acids are DNA, and RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Sci. Amer. 262:40, 1990). The invention embraces allantisense polynucleotides capable of inhibiting the production of CDSpolypeptide. In the cell, the antisense nucleic acids hybridize to thecorresponding mRNA, forming a double-stranded molecule. The antisensenucleic acids interfere with the translation of mRNA since the cellcannot translate mRNA that is double-stranded. Antisense oligomers ofabout 15 nucleotides are preferred, since they are easily synthesizedand are less likely to cause problems than larger molecules whenintroduced into the target CDS-producing cell. The use of antisensemethods to inhibit translation of genes is known (e.g., Marcus-Sakura,Anal. Biochem. 172:289, 1988).

[0074] In addition, ribozyme nucleotide sequences for CDS are includedin this invention. Ribozymes are hybrid RNA:DNA molecules possessing anability to specifically cleave other single-stranded RNA in a manneranalogous to DNA restriction endonucleases. Through the modification ofnucleotide sequences which encode such RNAs, it is possible to engineermolecules that recognize specific nucleotide sequences in an RNAmolecule and cleave it (Cech, J. Amer. Med. Assn. 260:3030, 1988). Anadvantage of this approach is that only mRNAs with particular sequencesare inactivated because they are sequence-specific.

[0075] The CDS DNA sequence may be inserted into an appropriaterecombinant expression vector. The term “recombinant expression vector”refers to a plasmid, virus or other vehicle that has been manipulated byinsertion or incorporation of the genetic sequences. Such expressionvectors contain a promoter sequence which facilitates efficienttranscription of the inserted genetic sequence in the host. Theexpression vector typically contains an origin of replication, apromoter, as well as specific genes which allow phenotypic selection ofthe transformed cells. Vectors suitable for use in the present inventioninclude, for example, vectors with a bacterial promoter and ribosomebinding site for expression in bacteria (Gold, Meth. Enzymol. 185:11,1990), expression vectors with mammalian or viral promoter and enhancerfor expression in mammalian cells (Kaufman, Meth. Enzymol. 185:487,1990) and baculovirus-derived vectors for expression in insect cells(Luckow et al., J. Virol. 67:4566, 1993). The DNA segment can be presentin the vector operably linked to regulatory elements, for example,constitutive or inducible promoters (e.g., T7, metallothionein I, CMV,or polyhedren promoters).

[0076] The vector may include a phenotypically selectable marker toidentify host cells which contain the expression vector. Examples ofmarkers typically used in prokaryotic expression vectors includeantibiotic resistance genes for ampicillin (β-lactamases), tetracyclineand chloramphenicol (chloramphenicol acetyltransferase). Examples ofsuch markers typically used in mammalian expression vectors include thegene for adenosine deaminase (ADA), aminoglycoside phosphotransferase(neo, G418), dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), andxanthine guanine phosphoriboseyltransferase (XGPRT, gpt).

[0077] In another preferred embodiment, the expression system used isone driven by the baculovirus polyhedrin promoter. The gene encoding thepolypeptide can be manipulated by standard techniques in order tofacilitate cloning into the baculovirus vector. A preferred baculovirusvector is the pBlueBac vector (Invitrogen, Sorrento, Calif.). The vectorcarrying the gene for the polypeptide is transfected into Spodopterafrugiperda (Sf9) cells by standard protocols, and the cells are culturedand processed to produce the recombinant polypeptide. See Summers etal., A Manual for Methods of Baculovirus Vectors and Insect Cell CultureProcedures, Texas Agricultural Experimental Station.

[0078] Once the entire coding sequence of the gene for the polypeptideshas been determined, the gene can be expressed in any number ofdifferent recombinant DNA expression systems to generate large amountsof polypeptide. Included within the present invention are polypeptideshaving native glycosylation sequences, and deglycosylated orunglycosylated polypeptides prepared by the methods described below.Examples of expression systems known to the skilled practitioner in theart include bacteria such as E. coli, yeast such as Pichia pastoris,baculovirus, and mammalian expression systems such as in COS or CHOcells.

[0079] The gene or gene fragment encoding the desired polypeptide can beinserted into an expression vector by standard subcloning techniques. Ina preferred embodiment, an E. coli expression vector is used whichproduces the recombinant protein as a fusion protein, allowing rapidaffinity purification of the protein. Examples of such fusion proteinexpression systems are the glutathione S-transferase system (Pharmacia,Piscataway, N.J.), the maltose binding protein system (NEB, Beverley,Mass.), the thiofusion system (Invotrogen, San Diego, Calif.), the FLAGsystem (IBI, New Haven, Conn.), and the 6xHis system (Qiagen,Chatsworth, Calif.). Some of these systems produce recombinantpolypeptides bearing only a small number of additional amino acids,which are unlikely to affect the CDS activity of the recombinantpolypeptide. For example, both the FLAG system and the 6xHis system addonly short sequences, both of which are known to be poorly antigenic andwhich do not adversely affect folding of the polypeptide to its nativeconformation. Other fusion systems produce proteins where it isdesirable to excise the fusion partner from the desired protein. In apreferred embodiment, the fusion partner is linked to the recombinantpolypeptide by a peptide sequence containing a specific recognitionsequence for a protease. Examples of suitable sequences are thoserecognized by the Tobacco Etch Virus protease (Life Technologies,Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.)or enterokinase (Invotrogen, San Diego, Calif.).

[0080] Production of Polypeptides

[0081] Polynucleotide sequences encoding CDS polypeptides of theinvention can be expressed in either prokaryotes or eukaryotes. Hostscan include microbial (bacterial), yeast, insect and mammalianorganisms. Methods of expressing DNA sequences inserted downstream ofprokaryotic or viral regulatory sequences in prokaryotes are known inthe art (Makrides, Microbio. Rev. 60:512, 1996). Biologically functionalviral and plasmid DNA vectors capable of expression and replication in aeukaryotic host are known in the art (Cachianes, Biotechniques 15:255,1993). Such vectors are used to incorporate DNA sequences of theinvention. DNA sequences encoding the inventive polypeptides can beexpressed in vitro by DNA transfer into a suitable host using knownmethods of transfection.

[0082] Sequences encoding CDS polypeptides may be inserted into arecombinant expression vector. The term “recombinant expression vector”refers to a plasmid, virus or other vehicle that has been manipulated byinserting or incorporating genetic sequences. Such expression vectorscontain a promoter sequence which facilitates efficient transcription ofthe inserted genetic sequence of the host. The expression vectortypically contains an origin of replication and a promoter, as well asspecific genes which allow phenotypic selection of the transformedcells. The DNA segment can be present in the vector, operably linked toregulatory elements, for example, a promoter (e.g., T7, metallothioneinI, or polyhedren promoters). Vectors suitable for use in the presentinvention include, for example, bacterial expression vectors, withbacterial promoter and ribosome binding sites, for expression inbacteria (Gold, Meth. Enzymol. 185:11, 1990), expression vector withanimal promoter and enhancer for expression in mammalian cells (Kaufman,Meth. Enzymol. 185:487, 1990) and baculovirus-derived vectors forexpression in insect cells (Luckow et al., J. Virol.67:4566, 1993).

[0083] The vector may include a phenotypically selectable marker toidentify host cells which contain the expression vector. Examples ofmarkers typically used in prokaryotic expression vectors includeantibiotic resistance genes for ampicillin (β-lactamases), tetracyclineand chloramphenicol (chloramphenicol acetyltransferase). Examples ofsuch markers typically used in mammalian expression vectors include thegene for adenosine deaminase (ADA), aminoglycoside phosphotransferase(neo, G418), dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (BPH), thymidine kinase (TK), andxanthine guanine phosphoriboseyltransferase (XGPRT, gpt).

[0084] In another preferred embodiment, the expression system used isone driven by the baculovirus polyhedrin promoter. The polynucleotideencoding CDS can be manipulated by standard techniques in order tofacilitate cloning into the baculovirus vector. See Ausubel et al.,supra. A preferred baculovirus vector is the pBlueBac vector(Invitrogen, Sorrento, Calif.). The vector carrying a polynucleotideencoding CDS is transfected into Spodoptera frugiperda (Sf9) cells bystandard protocols, and the cells are cultured and processed to producethe recombinant polypeptide. See Summers et al., A Manual for Methods ofBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station.

[0085] The polynucleotides of the present invention can be expressed inany number of different recombinant DNA expression systems to generatelarge amounts of polypeptide. Included within the present invention areCDS polypeptides having native glycosylation sequences, anddeglycosylated or unglycosylated polypeptides prepared by the methodsdescribed below. Examples of expression systems known to the skilledpractitioner in the art include bacteria such as E. coli, yeast such asPichia pastoris, baculovirus, and mammalian expression systems such asin Cos or CHO cells.

[0086] The polynucleotides of the present invention can be inserted intoan expression vector by standard subcloning techniques. In a preferredembodiment, an E. coli expression vector is used which produces therecombinant protein as a fusion protein, allowing rapid affinitypurification of the protein. Examples of such fusion protein expressionsystems are the glutathione S-transferase system (Pharmacia, Piscataway,N.J.), the maltose binding protein system (NEB, Beverley, Mass.), thethiofusion system (Invitrogen, San Diego, Calif.), the Strep-tag IIsystem (Genosys, Woodlands, Tex.), the FLAG system (IBI, New Haven,Conn.), and the 6xHis system (Qiagen, Chatsworth, Calif.). Some of thesesystems produce recombinant polypeptides bearing only a small number ofadditional amino acids, which are unlikely to affect the CDS ability ofthe recombinant polypeptide. For example, both the FLAG system and the6xHis system add only short sequences, both of which are known to bepoorly antigenic and which do not adversely affect folding of thepolypeptide to its native conformation. Other fusion systems produceproteins where it is desirable to excise the fusion partner from thedesired protein. In a preferred embodiment, the fusion partner is linkedto the recombinant polypeptide by a peptide sequence containing aspecific recognition sequence for a protease. Examples of suitablesequences are those recognized by the Tobacco Etch Virus protease (LifeTechnologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs,Beverley, Mass.) or enterokinase (Invitrogen, San Diego, Calif.).

[0087] In an embodiment of the present invention, the polynucleotidesencoding CDS are analyzed to detect putative transmembrane sequences.Such sequences are typically very hydrophobic and are readily detectedby the use of standard sequence analysis software, such as MacDNASIS(Hitachi, San Bruno, Calif.). The presence of transmembrane sequences isoften deleterious when a recombinant protein is synthesized in manyexpression systems, especially in E. coli, as it leads to the productionof insoluble aggregates which are difficult to renature into the nativeconformation of the polypeptide.

[0088] Accordingly, deletion of one or more of the transmembranesequences may be desirable. Deletion of transmembrane sequencestypically does not significantly alter the conformation or activity ofthe remaining polypeptide structure. However, one can determine whetherdeletion of one or more of the transmembrane sequences has effected thebiological activity of the CDS protein by, for example, assaying theactivity of the CDS protein containing one or more deleted sequences andcomparing this activity to that of unrnodified CDS. Examples of assaysfor CDS activity are described above.

[0089] Moreover, transmembrane sequences, being by definition embeddedwithin a membrane, are inaccessible as antigenic determinants to a hostimmune system. Antibodies to these sequences will not, therefore,provide immunity to the host and, hence, little is lost in terms ofgenerating monoclonal or polyclonal antibodies by omitting suchsequences from the recombinant polypeptides of the invention. Deletionof transmembrane-encoding sequences from the polynucleotide used forexpression can be achieved by standard techniques. See Ausubel et al.,zipra, Chapter 8. For example, fortuitously-placed restriction enzymesites can be used to excise the desired gene fragment, or the PCR can beused to amplify only the desired part of the gene.

[0090] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques. When the host is prokaryotic, such as E.coli, competent cells which are capable of DNA uptake can be preparedfrom cells harvested after exponential growth phases and subsequentlytreated by a CaCl₂ method using standard procedures. Alternatively,MgCl₂ or RbCl can be used. Transformation can also be performed afterforming a protoplast of the host cell or by electroporation.

[0091] When the host is a eukaryote, methods of transfection of DNA,such as calcium phosphate co-precipitates, conventional mechanicalprocedures, (e.g., microinjection), electroporation, liposome-encasedplasmids, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding CDS polypeptides of thepresent invention, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method uses a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus to transiently infect or transformeukaryotic cells and express the CDS polypeptides.

[0092] Expression vectors that are suitable for production of CDSpolypeptides preferably contain (1) prokaryotic DNA elements coding fora bacterial replication origin and an antibiotic resistance marker toprovide for the growth and selection of the expression vector in abacterial host; (2) eukaryotic DNA elements that control initiation oftranscription, such as a promoter; and (3) DNA elements that control theprocessing of transcripts, such as a transcriptiontermination/polyadenylation sequence. CDS polypeptides of the presentinvention preferably are expressed in eukaryotic cells, such asmammalian, insect and yeast cells. Mammalian cells are especiallypreferred eukaryotic hosts because mammalian cells provide suitablepost-translational modifications such as glycosylation. Examples ofmammalian host cells include Chinese hamster ovary cells (CHO-K1; ATCCCCL61), rat pituitary cells (GH₁; ATCC CCL82), HeLa S3 cells (ATCCCCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL1548) SV40-transformedmonkey kidney cells (COS-1; ATCC CRL 1650) and murine embryonic cells(NIH-3T3; ATCC CRL 1658). For a mammalian host, the transcriptional andtranslational regulatory signals may be derived from viral sources, suchas adenovirus, bovine papilloma virus, simian virus, or the like, inwhich the regulatory signals are associated with a particular gene whichhas a high level of expression. Suitable transcriptional andtranslational regulatory sequences also can be obtained from mammaliangenes, such as actin, collagen, myosin, and metallothionein genes.

[0093] Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273,1982); the TK promoterof Herpes virus (McKnight, Cell 31: 355, 1982); the SV40 early promoter(Benoist et al., Nature 290:304, 1981); the Rous sarcoma virus promoter(Gorman et al., Proc. Nat'l. Acad. Sci. USA 79:6777, 1982); and thecytomegalovirus promoter (Foecking et al., Gene 45:101, 1980).Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control fusion gene expression ifthe prokaryotic promoter is regulated by a eukaryotic promoter (Zhou etal., Mol. Cell. Biol. 10:4529, 1990; Kaufman et al, Nucl. Acids Res.19:4485, 1991).

[0094] An expression vector can be introduced into host cells using avariety of techniques including calcium phosphate transfection,liposome-mediated transfection, electroporation, and the like.Preferably, transfected cells are selected and propagated wherein theexpression vector is stably integrated in the host cell genome toproduce stable transformants. Techniques for introducing vectors intoeukaryotic cells and techniques for selecting stable transformants usinga dominant selectable marker are described, for example, by Ausubel andby Murray (ed.), Gene Transfer and Expression Protocols (Humana Press1991). Examples of mammalian host cells include COS, BHK, 293 and CHOcells.

[0095] Purification of Recombinant Polypeptides.

[0096] The polypeptide expressed in recombinant DNA expression systemscan be obtained in large amounts and tested for biological activity. Therecombinant bacterial cells, for example E. coli, are grown in any of anumber of suitable media, for example LB, and the expression of therecombinant polypeptide induced by adding IPTG to the media or switchingincubation to a higher temperature. After culturing the bacteria for afurther period of between 2 and 24 hours, the cells are collected bycentrifugation and washed to remove residual media. The bacterial cellsare then lysed, for example, by disruption in a cell homogenizer andcentrifuged to separate the dense inclusion bodies and cell membranesfrom the soluble cell components. This centrifugation can be performedunder conditions whereby the dense inclusion bodies are selectivelyenriched by incorporation of sugars such as sucrose into the buffer andcentrifugation at a selective speed. If the recombinant polypeptide isexpressed in the inclusion, these can be washed in any of severalsolutions to remove some of the contaminating host proteins, thensolubilized in solutions containing high concentrations of urea (e.g., 8M) or chaotropic agents such as guanidine hydrochloride in the presenceof reducing agents such as β-mercaptoethanol or DTT (dithiothreitol). Atthis stage it may be advantageous to incubate the polypeptide forseveral hours under conditions suitable for the polypeptide to undergo arefolding process into a conformation which more closely resembles thatof the native polypeptide. Such conditions generally include lowpolypeptide (concentrations less than 500 mg/ml), low levels of reducingagent, concentrations of urea less than 2 M and often the presence ofreagents such as a mixture of reduced and oxidized glutathione whichfacilitate the interchange of disulphide bonds within the proteinmolecule. The refolding process can be monitored, for example, bySDS-PAGE or with antibodies which are specific for the native molecule.Following refolding, the polypeptide can then be purified further andseparated from the refolding mixture by chromatography on any of severalsupports including ion exchange resins, gel permeation resins or on avariety of affinity columns.

[0097] Isolation and purification of host cell expressed polypeptide, orfragments thereof may be carried out by conventional means including,but not limited to, preparative chromatography and immunologicalseparations involving monoclonal or polyclonal antibodies.

[0098] These polypeptides may be produced in a variety of ways,including via recombinant DNA techniques, to enable large scaleproduction of pure, active CDS useful for screening compounds fortrilineage hematopoietic and anti-inflammatory therapeutic applications,and developing antibodies for therapeutic, diagnostic and research use.

[0099] Screening Assays using CDS Polypeptides

[0100] The CDS polypeptide of the present invention is useful in ascreening methodology for identifying compounds or compositions whichaffect cellular signaling of an inflammatory response. This methodcomprises incubating the CDS polypeptides or a cell transfected withcDNA encoding CDS, with a suitable substrate, for example, PA, underconditions sufficient to allow the components to interact, and thenmeasuring the effect of the compound or composition on CDS activity.See, for example, above, and Weeks et al., DNA Cell Biol. 16: 281-289,1997. The observed effect on CDS may be either inhibitory orstimulatory. Such compounds or compositions to be tested can be selectedfrom a combinatorial chemical library or any other suitable source(Hogan, Jr., Nat. Biotechnology 15:328, 1997).

[0101] Peptide Sequencing of Polypeptides

[0102] Substitutional variants typically contain the exchange of oneamino acid for another at one or more sites within the protein, and aredesigned to modulate one or more properties of the polypeptides such asstability against proteolytic cleavage. Substitutions preferably areconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of alanine to serine; arginine tolysine; asparigine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparigine; glutamate to aspartate;glycine to proline; histidine to asparigine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine,glutamine, or glutamate; methionine to leucine or isoleucine;phenylalanine to tyrosine, leucine or methionine; serine to threonine;threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan orphenylalanine; and valine to isoleucine or leucine. Insertional variantscontain fusion proteins such as those used to allow rapid purificationof the polypeptide and also can include hybrid polypeptides containingsequences from other proteins and polypeptides which are homologues ofthe inventive polypeptide. For example, an insertional variant couldinclude portions of the amino acid sequence of the polypeptide from onespecies, together with portions of the homologous polypeptide fromanother species. Other insertional variants can include those in whichadditional amino acids are introduced within the coding sequence of thepolypeptides. These typically are smaller insertions than the fusionproteins described above and are introduced, for example, to disrupt aprotease cleavage site.

[0103] Anti-CDS Antibodies

[0104] Antibodies to human CDS protein can be obtained using the productof a CDS expression vector or synthetic peptides derived from the CDScoding sequence coupled to a carrier (Pasnett et al., J. Biol. Chem.263:1728, 1988) as an antigen. The preparation of polyclonal antibodiesis well-known to those of sldl in the art. See, for example, Green etal., “Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992). Alternatively, a CDSantibody of the present invention may be derived as a rodent monoclonalantibody (MAb). Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art. See, for example,Kohler and Milstein, Nature 256:495, 1975, and Coligan et al. (eds.),Current Protocols in Immunology, 1:2.5.1-2.6.7 (John Wiley& Sons 1991).Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, verifying the presence of antibodyproduction by removing a serum sample, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

[0105] MAbs can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, 10:79-104 Humana Press, Inc. 1992. A CDS antibody of thepresent invention may also be derived from a subhuman primate. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., international patentpublication No. WO 91/11465 (1991), and in Losman et al., Int. J. Cancer46:310, 1990.

[0106] Alternatively, a therapeutically useful CDS antibody may bederived from a “humanized” monoclonal antibody. Humanized monoclonalantibodies are produced by transferring mouse complementaritydetermining regions from heavy and light chain variable regions of themouse antibody into a human antibody variable domain, and then,substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by the publication of Orlandi et al., Proc. Nat'l. Acad Sci.USA 86:3833, 1989. Techniques for producing humanized MAbs aredescribed, for example, by Jones et al., Nature 321:522, 1986; Riechmannet al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988;Carter et al., Proc. Nat'l Acad Sci. USA 89:4285, 1992; Sandhu, Crit.Rev. Biotech. 12: 437, 1992; and Singer et al., J. Immun. 150:2844,1993.

[0107] As an alternative, a CDS antibody of the present invention may bederived from human antibody fragments isolated from a combinatorialimmunoglobulin library. See, for example, Barbas et al., METHODS: ACompanion to Methods in Enzymology 2:119 1991, and Winter et al., Ann.Rev. Immunol. 12:433, 1994. Cloning and expression vectors that areuseful for producing a human immunoglobulin phage library can beobtained, for example, from STRATAGENE Cloning Systems (La Jolla,Calif.). In addition, a CDS antibody of the present invention may bederived from a human monoclonal antibody. Such antibodies are obtainedfrom transgenic mice that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain loci are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13, 1994; Lonberg et al.,Nature 368:856, 1994; and Taylor et al., Int. Immun. 6:579, 1994.

SEQUENCE LISTING

[0108] (2) INFORMATION FOR SEQ ID NO:1:

[0109] (i) SEQUENCE CHARACTERISTICS:

[0110] (a) LENGTH:2051

[0111] (b) TYPE: nucleic acid

1 19 1 2051 DNA Homo sapiens CDS (150)..(1532) 1 tctatggtgg ggccgcgttagtggctgcgg ctccgcggga ctccagggcg cggctgcgag 60 gtggcggggc gccccgcctgcagaaccctg cttgcagctc aggtttcggg gtgcttgagg 120 aggccgccac ggcagcgcgggagcggaag atg ttg gag ctg agg cac cgg gga 173 Met Leu Glu Leu Arg HisArg Gly 1 5 agc tgc ccc ggc ccc agg gaa gcg gtg tcg ccg cca cac cgc gaggga 221 Ser Cys Pro Gly Pro Arg Glu Ala Val Ser Pro Pro His Arg Glu Gly10 15 20 gag gcg gcc ggc ggc gac cac gaa acc gag agc acc agc gac aaa gaa269 Glu Ala Ala Gly Gly Asp His Glu Thr Glu Ser Thr Ser Asp Lys Glu 2530 35 40 aca gat att gat gac aga tat gga gat ttg gat tcc aga aca gat tct317 Thr Asp Ile Asp Asp Arg Tyr Gly Asp Leu Asp Ser Arg Thr Asp Ser 4550 55 gat att ccg gaa att cca cca tcc tca gat aga acc cct gag att ctc365 Asp Ile Pro Glu Ile Pro Pro Ser Ser Asp Arg Thr Pro Glu Ile Leu 6065 70 aaa aaa gct cta tct ggt tta tct tca agg tgg aaa aac tgg tgg ata413 Lys Lys Ala Leu Ser Gly Leu Ser Ser Arg Trp Lys Asn Trp Trp Ile 7580 85 cgt gga att ctc act cta act atg atc tcg ttg ttt ttc ctg atc atc461 Arg Gly Ile Leu Thr Leu Thr Met Ile Ser Leu Phe Phe Leu Ile Ile 9095 100 tat atg gga tcc ttc atg ctg atg ctt ctt gtt ctg ggc atc caa gtg509 Tyr Met Gly Ser Phe Met Leu Met Leu Leu Val Leu Gly Ile Gln Val 105110 115 120 aaa tgc ttc cat gaa att atc act ata ggt tat aga gtc tat cattct 557 Lys Cys Phe His Glu Ile Ile Thr Ile Gly Tyr Arg Val Tyr His Ser125 130 135 tat gat cta cca tgg ttt aga aca cta agt tgg tac ttt cta ttgtgt 605 Tyr Asp Leu Pro Trp Phe Arg Thr Leu Ser Trp Tyr Phe Leu Leu Cys140 145 150 gta aac tac ttt ttc tat gga gag act gta gct gat tat ttt gctaca 653 Val Asn Tyr Phe Phe Tyr Gly Glu Thr Val Ala Asp Tyr Phe Ala Thr155 160 165 ttt gtt caa aga gaa gaa caa ctt cag ttc ctc att cgc tac cataga 701 Phe Val Gln Arg Glu Glu Gln Leu Gln Phe Leu Ile Arg Tyr His Arg170 175 180 ttt ata tca ttt gcc ctc tat ctg gca ggt ttc tgc atg ttt gtactg 749 Phe Ile Ser Phe Ala Leu Tyr Leu Ala Gly Phe Cys Met Phe Val Leu185 190 195 200 agt ttg gtg aag gaa cat tat cgt ctg cag ttt tat atg ttcgca tgg 797 Ser Leu Val Lys Glu His Tyr Arg Leu Gln Phe Tyr Met Phe AlaTrp 205 210 215 act cat gtc act tta ctg ata act gtc act cag tca cac cttgtc atc 845 Thr His Val Thr Leu Leu Ile Thr Val Thr Gln Ser His Leu ValIle 220 225 230 caa aat ctg ttt gaa ggc atg ata tgg ttc ctt gtt cca atatca agt 893 Gln Asn Leu Phe Glu Gly Met Ile Trp Phe Leu Val Pro Ile SerSer 235 240 245 gtt atc tgc aat gac ata act gct tac ctt ttt gga ttt tttttt ggg 941 Val Ile Cys Asn Asp Ile Thr Ala Tyr Leu Phe Gly Phe Phe PheGly 250 255 260 aga act cca tta att aag ttg tct cct aaa aag act tgg gaagga ttc 989 Arg Thr Pro Leu Ile Lys Leu Ser Pro Lys Lys Thr Trp Glu GlyPhe 265 270 275 280 att ggt ggt ttc ttt tcc aca gtt gtg ttt gga ttc attgct gcc tat 1037 Ile Gly Gly Phe Phe Ser Thr Val Val Phe Gly Phe Ile AlaAla Tyr 285 290 295 gtg tta tcc aaa tac cag tac ttt gtc tgc cca gtg gaatac cga agt 1085 Val Leu Ser Lys Tyr Gln Tyr Phe Val Cys Pro Val Glu TyrArg Ser 300 305 310 gat gta aac tcc ttc gtg aca gaa tgt gag ccc tca gaactt ttc cag 1133 Asp Val Asn Ser Phe Val Thr Glu Cys Glu Pro Ser Glu LeuPhe Gln 315 320 325 ctt cag act tac tca ctt cca ccc ttt cta aag gca gtcttg aga cag 1181 Leu Gln Thr Tyr Ser Leu Pro Pro Phe Leu Lys Ala Val LeuArg Gln 330 335 340 gaa aga gtg agc ttg tac cct ttc cag atc cac agc attgca ctg tca 1229 Glu Arg Val Ser Leu Tyr Pro Phe Gln Ile His Ser Ile AlaLeu Ser 345 350 355 360 acc ttt gca tct tta att ggc cca ttt gga ggc ttcttt gct agt gga 1277 Thr Phe Ala Ser Leu Ile Gly Pro Phe Gly Gly Phe PheAla Ser Gly 365 370 375 ttc aaa aga gcc ttc aaa atc aag gat ttt gca aatacc att cct gga 1325 Phe Lys Arg Ala Phe Lys Ile Lys Asp Phe Ala Asn ThrIle Pro Gly 380 385 390 cat ggt ggg ata atg gac aga ttt gat tgt cag tatttg atg gca act 1373 His Gly Gly Ile Met Asp Arg Phe Asp Cys Gln Tyr LeuMet Ala Thr 395 400 405 ttt gta cat gtg tac atc aca agt ttt ata agg ggccca aat ccc agc 1421 Phe Val His Val Tyr Ile Thr Ser Phe Ile Arg Gly ProAsn Pro Ser 410 415 420 aaa gtg cta cag cag ttg ttg gtg ctt caa cct gaacag cag tta aat 1469 Lys Val Leu Gln Gln Leu Leu Val Leu Gln Pro Glu GlnGln Leu Asn 425 430 435 440 ata tat aaa acc ctg aag act cat ctc att gagaaa gga atc cta caa 1517 Ile Tyr Lys Thr Leu Lys Thr His Leu Ile Glu LysGly Ile Leu Gln 445 450 455 ccc acc ttg aag gta taactggatc cagagagggaaggactgaca agaaggaatt 1572 Pro Thr Leu Lys Val 460 attcagaaaa acactgacagatgttttata aattgtacag aaaaatagtt aaaaatgcaa 1632 taggttgaag ttttggagatatgtttctct ctgaaattac tgtgaatatt taacaaacac 1692 ttacttgatc tatgttatgaaataagtagc aaattgccag caaaatgtct tgtacctttt 1752 ctaaagtgta ttttctgatgtgaacttcct tccccttact tgctaggttt cataatttaa 1812 aagactggta tttaaaagagtcaaacacta taaaatgagt aagttgacga tgttttaaga 1872 ttgcacctgg cagtgtgcctttttgcacaa atatttactt ttgcacttgg agctgctttt 1932 aattttagca aaatgttttatgcaaggcac aataggaagt cagttctcct gcacttcctc 1992 ctcatgtagt ctggagtactttctaaaggg cttagttgga tttaaaaaaa aaaaaaaaa 2051 2 461 PRT Homo sapiens 2Met Leu Glu Leu Arg His Arg Gly Ser Cys Pro Gly Pro Arg Glu Ala 1 5 1015 Val Ser Pro Pro His Arg Glu Gly Glu Ala Ala Gly Gly Asp His Glu 20 2530 Thr Glu Ser Thr Ser Asp Lys Glu Thr Asp Ile Asp Asp Arg Tyr Gly 35 4045 Asp Leu Asp Ser Arg Thr Asp Ser Asp Ile Pro Glu Ile Pro Pro Ser 50 5560 Ser Asp Arg Thr Pro Glu Ile Leu Lys Lys Ala Leu Ser Gly Leu Ser 65 7075 80 Ser Arg Trp Lys Asn Trp Trp Ile Arg Gly Ile Leu Thr Leu Thr Met 8590 95 Ile Ser Leu Phe Phe Leu Ile Ile Tyr Met Gly Ser Phe Met Leu Met100 105 110 Leu Leu Val Leu Gly Ile Gln Val Lys Cys Phe His Glu Ile IleThr 115 120 125 Ile Gly Tyr Arg Val Tyr His Ser Tyr Asp Leu Pro Trp PheArg Thr 130 135 140 Leu Ser Trp Tyr Phe Leu Leu Cys Val Asn Tyr Phe PheTyr Gly Glu 145 150 155 160 Thr Val Ala Asp Tyr Phe Ala Thr Phe Val GlnArg Glu Glu Gln Leu 165 170 175 Gln Phe Leu Ile Arg Tyr His Arg Phe IleSer Phe Ala Leu Tyr Leu 180 185 190 Ala Gly Phe Cys Met Phe Val Leu SerLeu Val Lys Glu His Tyr Arg 195 200 205 Leu Gln Phe Tyr Met Phe Ala TrpThr His Val Thr Leu Leu Ile Thr 210 215 220 Val Thr Gln Ser His Leu ValIle Gln Asn Leu Phe Glu Gly Met Ile 225 230 235 240 Trp Phe Leu Val ProIle Ser Ser Val Ile Cys Asn Asp Ile Thr Ala 245 250 255 Tyr Leu Phe GlyPhe Phe Phe Gly Arg Thr Pro Leu Ile Lys Leu Ser 260 265 270 Pro Lys LysThr Trp Glu Gly Phe Ile Gly Gly Phe Phe Ser Thr Val 275 280 285 Val PheGly Phe Ile Ala Ala Tyr Val Leu Ser Lys Tyr Gln Tyr Phe 290 295 300 ValCys Pro Val Glu Tyr Arg Ser Asp Val Asn Ser Phe Val Thr Glu 305 310 315320 Cys Glu Pro Ser Glu Leu Phe Gln Leu Gln Thr Tyr Ser Leu Pro Pro 325330 335 Phe Leu Lys Ala Val Leu Arg Gln Glu Arg Val Ser Leu Tyr Pro Phe340 345 350 Gln Ile His Ser Ile Ala Leu Ser Thr Phe Ala Ser Leu Ile GlyPro 355 360 365 Phe Gly Gly Phe Phe Ala Ser Gly Phe Lys Arg Ala Phe LysIle Lys 370 375 380 Asp Phe Ala Asn Thr Ile Pro Gly His Gly Gly Ile MetAsp Arg Phe 385 390 395 400 Asp Cys Gln Tyr Leu Met Ala Thr Phe Val HisVal Tyr Ile Thr Ser 405 410 415 Phe Ile Arg Gly Pro Asn Pro Ser Lys ValLeu Gln Gln Leu Leu Val 420 425 430 Leu Gln Pro Glu Gln Gln Leu Asn IleTyr Lys Thr Leu Lys Thr His 435 440 445 Leu Ile Glu Lys Gly Ile Leu GlnPro Thr Leu Lys Val 450 455 460 3 38 PRT Drosophila 3 Lys Arg Ala PheLys Ile Lys Asp Phe Gly Asp Met Ile Pro Gly His 1 5 10 15 Gly Gly IleMet Asp Arg Phe Asp Cys Gln Phe Leu Met Ala Thr Phe 20 25 30 Val Asn ValTyr Ile Ser 35 4 38 PRT Homo sapiens 4 Lys Arg Ala Phe Lys Ile Lys AspPhe Ala Asn Thr Ile Pro Gly His 1 5 10 15 Gly Gly Ile Met Asp Arg PheAsp Cys Gln Tyr Leu Met Ala Thr Phe 20 25 30 Val His Val Tyr Ile Thr 355 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 5 cccaccatgg ccaggaatgg tatttgc 27 6 25 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 6 agtgatgtga attccttcgt gacag 25 7 29 DNA ArtificialSequence Description of Artificial Sequence Primer 7 ggctctagatattaatagta atcaattac 29 8 26 DNA Artificial Sequence Description ofArtificial Sequence Primer 8 cctcacgcat gcaccatggt aatagc 26 9 24 DNAArtificial Sequence Description of Artificial Sequence Primer 9ggtgcatgcg tgaggctccg gtgc 24 10 28 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 10 gtagttttca cggtacctga aatggaag 28 112488 DNA Homo sapiens CDS (25)..(1359) 11 cgacgtcggg ccgattttcc cagg atgaca gag ctg agg cag agg gtg gcc 51 Met Thr Glu Leu Arg Gln Arg Val Ala 15 cat gag ccg gtt gcg cca ccc gag gac aag gag tca gag tca gaa gca 99 HisGlu Pro Val Ala Pro Pro Glu Asp Lys Glu Ser Glu Ser Glu Ala 10 15 20 25aag gta gat gga gag act gca tcg gac agt gag agc cag gca gaa tcc 147 LysVal Asp Gly Glu Thr Ala Ser Asp Ser Glu Ser Gln Ala Glu Ser 30 35 40 gcaccc ctg cca gtc tct gca gat gat acc ccg gag gtc ctc aat agg 195 Ala ProLeu Pro Val Ser Ala Asp Asp Thr Pro Glu Val Leu Asn Arg 45 50 55 gcc ctttcc aac ttg tct tca aga tgg aag gac tgg tgg gtg aga ggc 243 Ala Leu SerAsn Leu Ser Ser Arg Trp Lys Asp Trp Trp Val Arg Gly 60 65 70 atc ctg actttg gcc atg att gca ttt ttc ttc atc atc att tac ctg 291 Ile Leu Thr LeuAla Met Ile Ala Phe Phe Phe Ile Ile Ile Tyr Leu 75 80 85 gga cca atg gttttg atg ata atc gtg atg tgc gtt cag att aag tgt 339 Gly Pro Met Val LeuMet Ile Ile Val Met Cys Val Gln Ile Lys Cys 90 95 100 105 ttc cat gagata atc act att ggc tac aac gtc tac cac tca tat gat 387 Phe His Glu IleIle Thr Ile Gly Tyr Asn Val Tyr His Ser Tyr Asp 110 115 120 ctg ccc tggttc agg acg ctc agc tgg tac ttt ctc ctg tgt gta aac 435 Leu Pro Trp PheArg Thr Leu Ser Trp Tyr Phe Leu Leu Cys Val Asn 125 130 135 tat ttc ttctat ggt gag aca gtg acg gat tac ttc ttc acc ctg gtc 483 Tyr Phe Phe TyrGly Glu Thr Val Thr Asp Tyr Phe Phe Thr Leu Val 140 145 150 cag aga gaagag cct ttg cgg att ctc agt aaa tac cac cgg ttc att 531 Gln Arg Glu GluPro Leu Arg Ile Leu Ser Lys Tyr His Arg Phe Ile 155 160 165 tcc ttt actctc tat cta ata gga ttc tgc atg ttt gta ctg agt ctg 579 Ser Phe Thr LeuTyr Leu Ile Gly Phe Cys Met Phe Val Leu Ser Leu 170 175 180 185 gtc aagaag cat tat cga ctg cag ttc tac atg ttt ggc tgg acc cat 627 Val Lys LysHis Tyr Arg Leu Gln Phe Tyr Met Phe Gly Trp Thr His 190 195 200 gtg acattg ctg att gtt gta aca cag tca cat ctt gtt atc cac aac 675 Val Thr LeuLeu Ile Val Val Thr Gln Ser His Leu Val Ile His Asn 205 210 215 cta tttgaa gga atg atc tgg ttc att gtc ccc ata tct tgt gtg atc 723 Leu Phe GluGly Met Ile Trp Phe Ile Val Pro Ile Ser Cys Val Ile 220 225 230 tgt aatgac atc atg gcc tat atg ttt ggc ttt ttc ttt ggt cgg acc 771 Cys Asn AspIle Met Ala Tyr Met Phe Gly Phe Phe Phe Gly Arg Thr 235 240 245 cca ctcatc aag ctg tcc ccg aag aag acc tgg gaa ggc ttc att ggg 819 Pro Leu IleLys Leu Ser Pro Lys Lys Thr Trp Glu Gly Phe Ile Gly 250 255 260 265 ggcttc ttt gct act gtg gtg ttt ggc ctt ctg ctg tcc tat gtg atg 867 Gly PhePhe Ala Thr Val Val Phe Gly Leu Leu Leu Ser Tyr Val Met 270 275 280 tccggg tac aga tgc ttt gtc tgc cct gtg gag tac aac aat gac acc 915 Ser GlyTyr Arg Cys Phe Val Cys Pro Val Glu Tyr Asn Asn Asp Thr 285 290 295 aacagc ttc act gtg gac tgt gag ccc tcg gac ctg ttt cgc ctg cag 963 Asn SerPhe Thr Val Asp Cys Glu Pro Ser Asp Leu Phe Arg Leu Gln 300 305 310 gagtac aac att cct ggg gtg atc cag tca gtc att ggc tgg aaa acg 1011 Glu TyrAsn Ile Pro Gly Val Ile Gln Ser Val Ile Gly Trp Lys Thr 315 320 325 gtccgg atg tac ccc ttc cag att cac agc atc gct ctc tcc acc ttt 1059 Val ArgMet Tyr Pro Phe Gln Ile His Ser Ile Ala Leu Ser Thr Phe 330 335 340 345gcc tcg ctc att ggc ccc ttt gga gga ttc ttc gca agt gga ttc aaa 1107 AlaSer Leu Ile Gly Pro Phe Gly Gly Phe Phe Ala Ser Gly Phe Lys 350 355 360cga gcc ttt aaa atc aaa gac ttt gcc aat acc att cct ggc cat gga 1155 ArgAla Phe Lys Ile Lys Asp Phe Ala Asn Thr Ile Pro Gly His Gly 365 370 375ggc atc atg gat cgc ttt gac tgc cag tat ctg atg gcc acc ttt gtc 1203 GlyIle Met Asp Arg Phe Asp Cys Gln Tyr Leu Met Ala Thr Phe Val 380 385 390aat gta tac atc gcc agt ttt atc aga ggc cct aac cca agc aaa ctg 1251 AsnVal Tyr Ile Ala Ser Phe Ile Arg Gly Pro Asn Pro Ser Lys Leu 395 400 405att cag cag ttc ctg act tta cgg cca gat cag cag ctc cac atc ttc 1299 IleGln Gln Phe Leu Thr Leu Arg Pro Asp Gln Gln Leu His Ile Phe 410 415 420425 aac acg ctg cgg tct cat ctg atc gac aaa ggg atg ctg aca tcc acc 1347Asn Thr Leu Arg Ser His Leu Ile Asp Lys Gly Met Leu Thr Ser Thr 430 435440 aca gag gac gag taggggccac ccagggccag gagaacagga acagaactga 1399 ThrGlu Asp Glu 445 gcaggggcag gtctccaagg caagcccagc tggtgtgact tagacaatgacgaggcttca 1459 actcactgtc tttttttttt tttttttttt ggagggtatt ttttatttgtgggttcaaaa 1519 aatctgtata tacagtctat gtgtttagaa tttgtgttgt aagtaaactacagctttgag 1579 ttggaaagaa gtcacgggtt gtaaaaccat ttggattttt ttaaaacaaaagtattaata 1639 atctggaaga cagtgttgcc caggtcagga gtgttttctt ggtggttccagcccccatca 1699 attgaactgt ttctgggctc agtcagacac agacattcat ctgtgtctgaccaaatcagg 1759 ggacttcccc acctgtggtg ggaggcacag cttagatgtt ttgtacacctggtcttttct 1819 agaaatccct gcttggagct gcagaagggt tgccttctgt aggtcggaggaatggaggct 1879 tactaaccag gtaagccttc tatgcatcca caccaaaatc ctgcagaatgtaagtaagct 1939 ctgctttata agatgggttc accttcatcg cagactgaaa gtttcagtttttattttttt 1999 cagaaagcac gaaaaattat ttataatagt ctggagaaaa aacacactgtaatatttcaa 2059 gtgtatgcag tagaatgtac tgtaactgag ccctttccca catgtctaggctccaatgtc 2119 tcctgtaggt ccacctaact gtgtgttttc agggacaatg ccatccatgtttgtgctgta 2179 gacttgctgc tgctgaatcc tttctgggga ctttctcatc gggcagggagcagagggctt 2239 ctcgttcatg caccctttgc ctgaacaccc atgtagctgc tgtgttgtgtatatattact 2299 cttaagagga gtgtgtgtgt ctgtgtttgt tttaaaagtc acttatttcttacagtgatt 2359 tcaattgcac catgacttct tcactaaaac cacaaagtcc tgcttaaaactatggaaaac 2419 ctaacctgat tagagccttg actattttga agattaaatg cacactttttatataaaaaa 2479 aaaaaaaaa 2488 12 445 PRT Homo sapiens 12 Met Thr GluLeu Arg Gln Arg Val Ala His Glu Pro Val Ala Pro Pro 1 5 10 15 Glu AspLys Glu Ser Glu Ser Glu Ala Lys Val Asp Gly Glu Thr Ala 20 25 30 Ser AspSer Glu Ser Gln Ala Glu Ser Ala Pro Leu Pro Val Ser Ala 35 40 45 Asp AspThr Pro Glu Val Leu Asn Arg Ala Leu Ser Asn Leu Ser Ser 50 55 60 Arg TrpLys Asp Trp Trp Val Arg Gly Ile Leu Thr Leu Ala Met Ile 65 70 75 80 AlaPhe Phe Phe Ile Ile Ile Tyr Leu Gly Pro Met Val Leu Met Ile 85 90 95 IleVal Met Cys Val Gln Ile Lys Cys Phe His Glu Ile Ile Thr Ile 100 105 110Gly Tyr Asn Val Tyr His Ser Tyr Asp Leu Pro Trp Phe Arg Thr Leu 115 120125 Ser Trp Tyr Phe Leu Leu Cys Val Asn Tyr Phe Phe Tyr Gly Glu Thr 130135 140 Val Thr Asp Tyr Phe Phe Thr Leu Val Gln Arg Glu Glu Pro Leu Arg145 150 155 160 Ile Leu Ser Lys Tyr His Arg Phe Ile Ser Phe Thr Leu TyrLeu Ile 165 170 175 Gly Phe Cys Met Phe Val Leu Ser Leu Val Lys Lys HisTyr Arg Leu 180 185 190 Gln Phe Tyr Met Phe Gly Trp Thr His Val Thr LeuLeu Ile Val Val 195 200 205 Thr Gln Ser His Leu Val Ile His Asn Leu PheGlu Gly Met Ile Trp 210 215 220 Phe Ile Val Pro Ile Ser Cys Val Ile CysAsn Asp Ile Met Ala Tyr 225 230 235 240 Met Phe Gly Phe Phe Phe Gly ArgThr Pro Leu Ile Lys Leu Ser Pro 245 250 255 Lys Lys Thr Trp Glu Gly PheIle Gly Gly Phe Phe Ala Thr Val Val 260 265 270 Phe Gly Leu Leu Leu SerTyr Val Met Ser Gly Tyr Arg Cys Phe Val 275 280 285 Cys Pro Val Glu TyrAsn Asn Asp Thr Asn Ser Phe Thr Val Asp Cys 290 295 300 Glu Pro Ser AspLeu Phe Arg Leu Gln Glu Tyr Asn Ile Pro Gly Val 305 310 315 320 Ile GlnSer Val Ile Gly Trp Lys Thr Val Arg Met Tyr Pro Phe Gln 325 330 335 IleHis Ser Ile Ala Leu Ser Thr Phe Ala Ser Leu Ile Gly Pro Phe 340 345 350Gly Gly Phe Phe Ala Ser Gly Phe Lys Arg Ala Phe Lys Ile Lys Asp 355 360365 Phe Ala Asn Thr Ile Pro Gly His Gly Gly Ile Met Asp Arg Phe Asp 370375 380 Cys Gln Tyr Leu Met Ala Thr Phe Val Asn Val Tyr Ile Ala Ser Phe385 390 395 400 Ile Arg Gly Pro Asn Pro Ser Lys Leu Ile Gln Gln Phe LeuThr Leu 405 410 415 Arg Pro Asp Gln Gln Leu His Ile Phe Asn Thr Leu ArgSer His Leu 420 425 430 Ile Asp Lys Gly Met Leu Thr Ser Thr Thr Glu AspGlu 435 440 445 13 2103 DNA Homo sapiens 13 tctatggtgg ggccgcgttagtggctgcgg ctccgcggga ctccagggcg cggctgcgag 60 gtggcggggc gccccgcctgcagaaccctg cttgcagctc aggtttcggg gtgcttgagg 120 aggccgccac ggcagcgcgggagcggaaga tgttggagct gaggcaccgg ggaagctgcc 180 ccggccccag ggaagcggtgtcgccgccac accgcgaggg agaggcggcc ggcggcgacc 240 acgaaaccga gagcaccagcgacaaagaaa cagatattga tgacagatat ggagatttgg 300 attccagaac agattctgatattccggaaa ttccaccatc ctcagataga acccctgaga 360 ttctcaaaaa agctctatctggtttatctt caaggtggaa aaactggtgg atacgtggaa 420 ttctcactct aactatgatctcgttgtttt tcctgatcat ctatatggga tccttcatgc 480 tgatgcttct tgttctgggcatccaagtga aatgcttcca tgaaattatc actataggtt 540 atagagtcta tcattcttatgatctaccat ggtttagaac actaagttgg tactttctat 600 tgtgtgtaaa ctactttttctatggagaga ctgtagctga ttattttgct acatttgttc 660 aaagagaaga acaacttcagttcctcattc gctaccatag atttatatca tttgccctct 720 atctggcagg tttctgcatgtttgtactga gtttggtgaa ggaacattat cgtctgcagt 780 tttatatgtt cgcatggactcatgtcactt tactgataac tgtcactcag tcacaccttg 840 tcatccaaaa tctgtttgaaggcatgatat ggttccttgt tccaatatca agtgttatct 900 gcaatgacat aactgcttacctttttggat ttttttttgg gagaactcca ttaattaagt 960 tgtctcctaa aaagacttgggaaggattca ttggtggttt cttttccaca gttgtgtttg 1020 gattcattgc tgcctatgtgttatccaaat accagtactt tgtctgccca gtggaatacc 1080 gaagtgatgt aaactccttcgtgacagaat gtgagccctc agaacttttc cagcttcaga 1140 cttactcact tccaccctttctaaaggcag tcttgagaca ggaaagagtg agcttgtacc 1200 ctttccagat ccacagcattgcactgtcaa cctttgcatc tttaattggc ccatttggag 1260 gcttctttgc tagtggattcaaaagagcct tcaaaatcaa ggattttgca aataccattc 1320 ctggacatgg tgggataatggacagatttg attgtcagta tttgatggca acttttgtac 1380 atgtgtacat cacaagttttataaggggcc caaatcccag caaagtgcta cagcagttgt 1440 tggtgcttca acctgaacagcagttaaata tatataaaac cctgaagact catctcattg 1500 agaaaggaat cctacaacccaccttgaagg tataactgga tccagagagg gaaggactga 1560 caagaaggaa ttattcagaaaaacactgac agatgtttta taaattgtac agaaaaatag 1620 ttaaaaatgc aataggttgaagttttggag atatgtttct ctctgaaatt actgtgaata 1680 tttaacaaac acttacttgatctatgttat gaaataagta gcaaattgcc agcaaaatgt 1740 cttgtacctt ttctaaagtgtattttctga tgtgaacttc cttcccctta cttgctaggt 1800 ttcataattt aaaagactggtatttaaaag agtcaaacac tataaaatga gtaagttgac 1860 gatgttttaa gattgcacctggcagtgtgc ctttttgcac aaatatttac ttttgcactt 1920 ggagctgctt ttaattttagcaaaatgttt tatgcaaggc acaataggaa gtcagttctc 1980 ctgcacttcc tcctcatgtagtctggagta ctttctaaag ggcttagttg gatttaaaaa 2040 aaaaaaaaaa agggcggccgctctagagga tccctcgagg ggcccaagct tacgcgtgca 2100 tgc 2103 14 45 PRT Homosapiens 14 Gln Ser His Leu Val Ile His Asn Leu Phe Glu Gly Met Ile TrpPhe 1 5 10 15 Ile Val Pro Ile Ser Cys Val Ile Cys Asn Asp Ile Met AlaTyr Met 20 25 30 Phe Gly Phe Phe Phe Gly Arg Thr Pro Leu Ile Lys Leu 3540 45 15 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 15 aggacgcata tgagtggtag ac 22 16 21 DNAArtificial Sequence Description of Artificial Sequence Primer 16gactctagcc taggcttttg c 21 17 249 PRT E. coli 17 Met Leu Ala Ala Trp GluTrp Gly Gln Leu Ser Gly Phe Thr Thr Arg 1 5 10 15 Ser Gln Arg Val TrpLeu Ala Val Leu Cys Gly Leu Leu Leu Ala Leu 20 25 30 Met Leu Phe Leu LeuPro Glu Tyr His Arg Asn Ile His Gln Pro Leu 35 40 45 Val Glu Ile Ser LeuTrp Ala Ser Leu Gly Trp Trp Ile Val Ala Leu 50 55 60 Leu Leu Val Leu PheTyr Pro Gly Ser Ala Ala Ile Trp Arg Asn Ser 65 70 75 80 Lys Thr Leu ArgLeu Ile Phe Gly Val Leu Thr Ile Val Pro Phe Phe 85 90 95 Trp Gly Met LeuAla Leu Arg Ala Trp His Tyr Asp Glu Asn His Tyr 100 105 110 Ser Gly AlaIle Trp Leu Leu Tyr Val Met Ile Leu Val Trp Gly Ala 115 120 125 Asp SerGly Ala Tyr Met Phe Gly Lys Leu Phe Gly Lys His Lys Leu 130 135 140 AlaPro Lys Val Ser Pro Gly Lys Thr Trp Gln Gly Phe Ile Gly Gly 145 150 155160 Leu Ala Thr Ala Ala Val Ile Ser Trp Gly Tyr Gly Met Trp Ala Asn 165170 175 Leu Asp Val Ala Pro Val Thr Leu Leu Ile Cys Ser Ile Val Ala Ala180 185 190 Leu Ala Ser Val Leu Gly Asp Leu Thr Glu Ser Met Phe Lys ArgGlu 195 200 205 Ala Gly Ile Lys Asp Ser Gly His Leu Ile Pro Gly His GlyGly Ile 210 215 220 Leu Asp Arg Ile Asp Ser Leu Thr Ala Ala Val Pro ValPhe Ala Cys 225 230 235 240 Leu Leu Leu Leu Val Phe Arg Thr Leu 245 18457 PRT Yeast 18 Met Ser Asp Asn Pro Glu Met Lys Pro His Gly Thr Ser LysGlu Ile 1 5 10 15 Val Glu Ser Val Thr Asp Ala Thr Ser Lys Ala Ile AspLys Leu Gln 20 25 30 Glu Glu Leu His Lys Asp Ala Ser Glu Ser Val Thr ProVal Thr Lys 35 40 45 Glu Ser Thr Ala Ala Thr Lys Glu Ser Arg Lys Tyr AsnPhe Phe Ile 50 55 60 Arg Thr Val Trp Thr Phe Val Met Ile Ser Gly Phe PheIle Thr Leu 65 70 75 80 Ala Ser Gly His Ala Trp Cys Ile Val Leu Ile LeuGly Cys Gln Ile 85 90 95 Ala Thr Phe Lys Glu Cys Ile Ala Val Thr Ser AlaSer Gly Arg Glu 100 105 110 Lys Asn Leu Pro Leu Thr Lys Thr Leu Asn TrpTyr Leu Leu Phe Thr 115 120 125 Thr Ile Tyr Tyr Leu Asp Gly Lys Ser LeuPhe Lys Phe Phe Gln Ala 130 135 140 Thr Phe Tyr Glu Tyr Pro Val Leu AsnPhe Ile Val Thr Asn His Lys 145 150 155 160 Phe Ile Cys Tyr Cys Leu TyrLeu Met Gly Phe Val Leu Phe Val Cys 165 170 175 Ser Leu Arg Lys Gly PheLeu Lys Phe Gln Phe Gly Ser Leu Cys Val 180 185 190 Thr His Met Val LeuLeu Leu Val Val Phe Gln Ala His Leu Ile Ile 195 200 205 Lys Asn Val LeuAsn Gly Leu Phe Trp Phe Leu Leu Pro Cys Gly Leu 210 215 220 Val Ile ValAsn Asp Ile Phe Ala Tyr Leu Cys Gly Ile Thr Phe Gly 225 230 235 240 LysThr Lys Leu Ile Glu Ile Ser Pro Lys Lys Thr Leu Glu Gly Phe 245 250 255Leu Gly Ala Trp Phe Phe Thr Ala Leu Ala Ser Ile Ile Leu Thr Arg 260 265270 Ile Leu Ser Pro Tyr Thr Tyr Leu Thr Cys Pro Val Glu Asp Leu His 275280 285 Thr Asn Phe Phe Ser Asn Leu Thr Cys Glu Leu Asn Pro Val Phe Leu290 295 300 Pro Gln Val Tyr Arg Leu Pro Pro Ile Phe Phe Asp Lys Val GlnIle 305 310 315 320 Asn Ser Ile Thr Val Lys Pro Ile Tyr Phe His Ala LeuAsn Leu Ala 325 330 335 Thr Phe Ala Ser Leu Phe Ala Pro Phe Gly Gly PhePhe Ala Ser Gly 340 345 350 Leu Lys Arg Thr Phe Lys Val Lys Asp Phe GlyHis Ser Ile Pro Gly 355 360 365 His Gly Gly Ile Thr Asp Arg Val Asp CysGln Phe Ile Met Gly Ser 370 375 380 Phe Ala Asn Leu Tyr Tyr Glu Thr PheIle Ser Glu His Arg Ile Thr 385 390 395 400 Val Asp Thr Val Leu Ser ThrIle Leu Met Asn Leu Asn Asp Lys Gln 405 410 415 Ile Ile Glu Leu Ile AspIle Leu Ile Arg Phe Leu Ser Lys Lys Gly 420 425 430 Ile Ile Ser Ala LysAsn Phe Glu Lys Leu Ala Asp Ile Phe Asn Val 435 440 445 Thr Lys Lys SerLeu Thr Asn His Ser 450 455 19 446 PRT Drosophila 19 Met Ala Glu Val ArgArg Arg Lys Gly Glu Asp Glu Pro Leu Glu Asp 1 5 10 15 Thr Ala Ile SerGly Ser Asp Ala Ala Asn Lys Arg Asn Ser Ala Ala 20 25 30 Asp Ser Ser AspHis Val Asp Ser Glu Glu Glu Lys Ile Pro Glu Glu 35 40 45 Lys Phe Val AspGlu Leu Ala Lys Asn Leu Pro Gln Gly Thr Asp Lys 50 55 60 Thr Pro Glu IleLeu Asp Ser Ala Leu Lys Asp Leu Pro Asp Arg Trp 65 70 75 80 Lys Asn TrpVal Ile Arg Gly Ile Phe Thr Trp Ile Met Ile Cys Gly 85 90 95 Phe Ala LeuIle Ile Tyr Gly Gly Pro Leu Ala Leu Met Ile Thr Thr 100 105 110 Leu LeuVal Gln Val Lys Cys Phe Gln Glu Ile Ile Ser Ile Gly Tyr 115 120 125 GlnVal Tyr Arg Ile His Gly Leu Pro Trp Phe Arg Ser Leu Ser Trp 130 135 140Tyr Phe Leu Leu Thr Ser Asn Tyr Phe Phe Tyr Gly Glu Asn Leu Val 145 150155 160 Asp Tyr Phe Gly Val Val Ile Asn Arg Val Glu Tyr Leu Lys Phe Leu165 170 175 Val Thr Tyr His Arg Phe Leu Ser Phe Ala Leu Tyr Ile Ile GlyPhe 180 185 190 Val Trp Phe Val Leu Ser Leu Val Lys Lys Tyr Tyr Ile LysGln Phe 195 200 205 Ser Leu Phe Ala Trp Thr His Val Ser Leu Leu Ile ValVal Thr Gln 210 215 220 Ser Tyr Leu Ile Ile Gln Asn Ile Phe Glu Gly LeuIle Trp Phe Ile 225 230 235 240 Val Pro Val Ser Met Ile Val Cys Asn AspVal Met Ala Tyr Val Phe 245 250 255 Gly Phe Phe Phe Gly Arg Thr Pro LeuIle Lys Leu Ser Pro Lys Lys 260 265 270 Thr Trp Glu Gly Phe Ile Gly GlyGly Phe Ala Thr Val Leu Phe Gly 275 280 285 Ile Leu Phe Ser Tyr Val LeuCys Asn Tyr Gln Tyr Phe Ile Cys Pro 290 295 300 Ile Gln Tyr Ser Glu GluGln Gly Arg Met Thr Met Ser Cys Val Pro 305 310 315 320 Ser Tyr Leu PheThr Pro Gln Glu Tyr Ser Leu Lys Leu Phe Gly Ile 325 330 335 Gly Lys ThrLeu Asn Leu Tyr Pro Phe Ile Trp His Ser Ile Ser Leu 340 345 350 Ser LeuPhe Ser Ser Ile Ile Gly Pro Phe Gly Gly Phe Phe Ala Ser 355 360 365 GlyPhe Lys Arg Ala Phe Lys Ile Lys Asp Phe Gly Asp Met Ile Pro 370 375 380Gly His Gly Gly Ile Met Asp Arg Phe Asp Cys Gln Phe Leu Met Ala 385 390395 400 Thr Phe Val Asn Val Tyr Ile Ser Phe Ile Arg Thr Pro Ser Pro Ala405 410 415 Lys Leu Leu Thr Gln Ile Tyr Asn Leu Lys Pro Asp Gln Gln TyrGln 420 425 430 Ile Tyr Gln Ser Leu Lys Asp Asn Leu Gly His Met Leu Thr435 440 445

We claim:
 1. An isolated polynucleotide, comprising a polynucleotidesequence selected from the group consisting of: (a) the DNA sequence ofFIG. 8 and biologically active fragments thereof; and (b) a DNA sequencewhich encodes the polypeptide of FIG. 8 and biologically activefragments thereof; wherein said polynucleotide encodes a polypeptidehaving CDP-diacylglycerol synthase (CDS) activity.
 2. A method ofexpressing a polypeptide from the polynucleotide of claim 1, comprising:(a) introducing into a cell the polynucleotide of claim 1, wherein saidpolynucleotide is operably linked to a promoter; and (b) maintaining orgrowing said cell under conditions that result in the expression of apolypeptide having CDS activity.
 3. The isolated polynucleotide of claim1, wherein said polynucleotide comprises (a) the DNA sequence of FIG. 8or (b) the DNA sequence encoding the polypeptide of FIG.
 8. 4. Theisolated polynucleotide of claim 3, wherein said polynucleotidecomprises the DNA sequence of FIG.
 8. 5. The isolated polynucleotide ofclaim 3, wherein said polynucleotide comprises the DNA sequence encodingthe polypeptide of FIG.
 8. 6. An isolated polynucleotide, comprising apolynucleotide sequence that hybridizes under high stringency conditionsto the DNA sequence selected from the group consisting of: (i) thecomplement of the coding region of the DNA sequence of (a) of claim 3;and (ii) the complement of the DNA sequence of claim (b) of claim 3;wherein said polynucleotide encodes a protein having CDS activity.
 7. Anisolated polynucleotide, comprising a polynucleotide sequence having atleast 85 percent identity to the DNA sequence selected from the groupconsisting of: (i) the coding region of the DNA sequence of (a) of claim3; and (ii) the DNA sequence of (b) of claim 3; wherein saidpolynucleotide encodes a protein having CDS activity.
 8. The isolatedpolynucleotide of claim 1, wherein said polynucleotide encodes apolypeptide having an intact CDS N-terminal region.
 9. An isolatedpolypeptide, comprising the amino acid sequence of FIG. 8 andbiologically active fragments thereof, wherein said polypeptide has CDSactivity.
 10. The isolated polypeptide of claim 9, wherein saidpolypeptide comprises the amino acid sequence of FIG.
 8. 11. An isolatedpolypeptide, comprising an amino acid sequence having 85 percentsequence identity to the amino acid sequence of FIG. 8, wherein saidpolypeptide has CDS activity.
 12. An isolated polypeptide, comprisingthe polypeptide encoded by the polynucleotide of claim
 6. 13. Anisolated polypeptide, comprising the polypeptide encoded by thepolynucleotide of claim
 7. 14. A method for screening one or morecompounds to determine whether said one or more compounds increases ordecreases CDS activity, comprising: (a) contacting the polypeptide ofclaim 9 with one or more substrates for said polypeptide and with saidone or more compounds; and (b) measuring whether the CDS activity ofsaid polypeptide is increased or decreased by said one or morecompounds.
 15. The method of claim 14, wherein said one or morecompounds is selected from a combinatorial chemical library.
 16. Themethod of claim 9, wherein said polypeptide contains an intact CDSN-terminal region.